FACTORS AFFECTING THE PRODUCTION OF DEXTRAN HAVING PHYSICO-CHEMICAL PROPERTIES OF

A PLASMA VOLUME EXPANDER

DISSERTATION

Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

MOSTAFA KAMAL HAMDY, B. Sc., M.Sc. i'

The Ohio State University

1953

Approved by: 1

FACTORS AFFECTING THE PRODUCTION OF DEXTRAN HAVING PHYSICO-CHEMICAL PROPERTIES OF A PLASMA VOLUME EXPANDER

INTRODUCTION

During the second World War, it was impossible to supply the demand for blood plasma. This stimulated a great number of investigators to study the problem of blood expanders with the aim of developing a synthetic or naturally occurring sub­ stance for this use.

In general, a good plasma extender must be of the pro­ per molecular size, homogeneous, non-toxic, non-antigenic, soluble in dilute salt solutions, and neutral in reaction. It must also be stable so that it can be used under a vari­ ety of geographical and climatic conditions.

With the potential possibilities of widespread casual­ ties, because of the use of atomic weapons, civilian defense authorities advise that to be properly prepared, a stock pile of at least one pint of plasma for every person in metropoli­ tan areas is needed. Without this stock pile it is estimated that all survivors of such an attack would have to be bled to furnish enough blood fluids for the injured. In addition to the advantages derived from a stable material for intravenous use, it would be possible to eliminate the danger of infect- 91 that the rate of degradation is directly proportional to the intensity of the ultrasonic vibrations, as presented in table 3 0 .

Molecular weight distribution. A large amount of high molecular weight dextran B - % 1 2 was subjected to ultrasonic vibrations, under controlled conditions, to reach a value of O.2I4.-O.26 intrinsic viscosity”. The degraded polymer was subjected to molecular weight homogeneity test, as formerly described, using two liters of 2 per cent aqueous solution. Five main fractions (U^-U^) were obtained at the following isopropyl alcohol concentrations: 32.5, 37*5* 1+2.and 55>*0 per cents. Table 31 presents the data for the per cent yield and the intrinsic viscosity of the fractions, indicat­ ing that 91+.28 per cent of the degraded dextran are in the range of the proper intrinsic viscosity of a plasma extender, and that 9 2 .2 9 per cent of the fractionated polymer has al­ most the same intrinsic viscosity of the original degraded material. Further fractionation was carried out on fractions U-j_ and Ug* to separate each of them to the subfractions a, b, c, etc., and the data for percentage yields of the sub­ fractions (based on the original degraded dextran) and their

intrinsic viscosity are recorded in table 3 2 , from which the following conclusions may be drawn.

This degradation was carried out in Battelle Memorial In­ stitute, using a Radio Sonorator Model RS-2 . 92

TABUS 30

Effect of calculated energy output (intensity) of the ultrasonic'''* vibrations on the degradation of dextran B-512

Total period ^ sp/ c of energy output irradiation J-l-30 watts 630 watts

. 0 min. 2.92 2.92 30 " 1.66 1.65 60 " 1.11+ 1.05 90 " 0.91+ 0.85 120 " 0.87 0.65 150 ” 0.72 0.50

18 0 11 0.54 0.1+2 210 " 0.50 0.38

Energy output ■ voltage input x current input watts k.v. m. amps 93

TABLE 31

Fractionation of two liters of a 2 per cent aqueous solution of ultrasonic depolymerized dextran B-512*

W W Alcohol Weight of fo Notation 4\ ml. fractions yield of t n g. fraction

481.46 27.71 69.27 ui 0.260 108.00 4.59 II.49 0.255 121}.. 30 0.250 3.14 7.87 U3 11+4.30 1 .4 6 3.66 \ 0.240 275.00 1.99 0.190 0.79 US

This dextran has an intrinsic viscosity value of 0 . 24- 0 . 2 6 . Percentage yield based on the original weight of degraded dextran. Intrinsic viscosity measurements were made at 25 C t o.o5 . TABLE 32

The molecular weight distribution of ultrasonic degraded dextran B-5 1 2

Fraction Subfraction % IPA yield ft]

u i a 32.5 38.36 0.280 b 35.0 114-.62 0.275 c 37.5 5.99 0.270 d i+2.5 1.83 0.230 e 55.0 1.66 0.200

V2 a 37.5 6.25 0.270 b ll-O.O 3.16 0 .18 0 c 55.0 0.99 0 .1 5 0

Percentage yield based on the original weight of dextran before fractiohation. 95 (1 ) Most of the fractions were precipitated in the first and second subfractions.

(2 ) The intrinsic viscosity for the majority of the subfractions, is in the range of the desired molecular size.

(3 ) A narrow range in molecular weight distribution is present in the ultrasonic degraded dextran.

Comparison of Degradation Processes

In order to provide a good comparison of the various methods for the degradation of high molecular weight dex­ tran to the desired molecular size, the same batch of dex­ tran B-512 was used in this study. A 6 per cent aqueous solution of this dextran was hy­ drolyzed using 0 .10N hydrochloric acid at 5 5 -6 0 C in a wa­ ter bath until an intrinsic viscosity value 0 .1 9-0 .2 0 was reached. Another 6 per cent aqueous solution from the same batch was depolymerized by ultrasonic vibrations until an intrinsic viscosity of O.2I4.-O.26 was reached. The depolymer­ ization in both methods was followed by viscosity measure­ ments. The results are presented in table 33 &n<3- in figure 11, which indicate that the fall in viscosity was gradual in the case of ultrasonic degradation, in contrast to that of the acid hydrolysis, which fell rapidly. The third sam­ ple was degraded through autolysis (extended incubation of the fermented media). The autolyzed dextran had an intrin- 96

TABLE 33

Comparison of acid and ultrasonic depolymerization of the high, molecular -weight dextran

Time of hydrolysis Relative viscosity of the 6% solution in hours Acid Ultrasonic hydrolysis depolymerization

0 .0 2 3 .0 0 2 3 .0 0 0.5 10.77 1 8 .7 0 1 .0 7.^6 16.15 1.5 6.15 1 3 .8 0 2 .0 5.38 12.30 2.5 I4-. 60 11.60 3.0 Ij.. 18 IO.J4.O 3.5 3.77 10.07 k-o 3.07 9 .2 0 1J-.5 2.75 8.77 5.0 ------8 .2 3 5.5 7.69 6.0 7 -J+8 6.5 7.23 7.0 7.07 7.5 6.92 8.0 6 .7 6 8.5 6.23 9.0

Viscosity measurements were made at 25 C. 'i/e v/scoJ/'fy of 6 % so/af/0/2^. / Figure I I Comparison, between add and Figure Comparison, II add between 2 ih oeua wih dextran. weight molecular a onhigh degradation ultrasonic 3 Time Time 4 inhours. S 6 7

Q 9 98 sic viscosity of 0.33 ^ 0.05.

Two liters of 2 per cent aqueous solution of each, de­ graded polymer were subjected to molecular weight homogene­ ity tests, as previously described. Table 3k- depicts the yield of the fractions and their intrinsic viscosity for the three different degraded polymers. Further fractionation was carried on some of these fractions to separate them to subfractions, a, b, c, etc. Intrinsic viscosity measurements were made on all the subfractions, while light-scattering ex­ periments for molectilar weight determinations were performed on some of the fractions and their subfractions. The results of these measurements are recorded in tables 18, 32 and 35 from which the following conclusions can be attained:

(1 ) Acid and autolysis degradation lead to a very high polydispersed polymer.

(2 ) Ultrasonic depolymerization of dextran results in a product which is more uniform in respect to molecular weight and is in the range of the value desired for use as a plasma volume expander.

Integral distribution curves. The integral distribu­ tion curve provides a simple and direct means for comparing the mo'lecular weight or intrinsic viscosity distributions produced by the different procedures of degradation under investigation. Using the dry weights and the intrinsic viscosities of the subfractions obtained from fractional 99

TABLE 34

Fractional precipitation of two liters of 2 per cent aqueous solution of autolyzed, acid hydrolyzed and ultrasonieally depolymerized dextran B-512

% Autolyzed Acid hydrolyzed Ultrasonieally IPA % % degraded yield m i yield HI Jo yield

A 32.5 49.52 0.48 — 69.27 0. 260 35.0 --- — 25.35 0.30 ------37.5 14.52 0.36 --- — 11.49 0.255

4 0 .0 --- — 21.85 0.19 ------

4 2 .5 6.82 0 .27 --- — 7.87 0.250 45. o --- — 23.62 0.16 ------

47.5 5.87 0.18 --- — 3.66 0.240 5o.o 3.97 0 .1 4 10.25 0.11 1.99 0.190

55.0 — 5.50 0.09 --- ———

Sample not recovered. TABLE 35

The molecular weight distribution of acid hydrolyzed dextran B-512

Fraction % Subr % Wt.-ave. vr IPA fraction yield r m Mol. wt.

32.5 a 7.50 0.38 205,000 G1JL 35.0 b 9.22 0.30 127,800 40.0 c 6.05 0.22 69,000 11-5 .0 d 1.12 0.19 55,500 30.0 e ----- — a 55.0 f 7.50 0.16 36,000 G 2 37.5 a 13.50 0.28 111,000 5.0.0 b 1.12 0.21 63,000 1+2.5 c 2.25 0.18 46,000 1+5 . o d 1.37 0.15 32,000 5o.o e o.5o 0.12 20,000 5 5 .o f 1.37 0.08 9,000 Go 40.0 a 9.50 0.17 41,000 42.5 b 6.00 0.15 32,000 5o.o c 2.25 0.12 20,000 55.0 d 2.62 0.06 51,000

G u 4 2 .5 a 4.00 0.14 28,000 + 4 5 .0 b 1.37 0.11 18,000 5o.o c 1.62 0.09 12,000 5 5 .o d 1.37 0.04 2,500

Weight-average molecular weight as measured by light- scattering. No sample was recovered. 2 ing debilitated patients through.the use of infected blood products. It must be made clear that these blood expanders serve only as a temporary blood substitute; they cannot fulfill the immunological, nutritive, or buffering function of the natu­ ral plasma, as reported by Bull et al (1914-9). At present there is no substitute for red blood cells. The sole func­ tion of synthetic expanders in transfusion is the maintenance of blood volume, to lessen the possibility of oligemic shock or heart failure.

Bayliss (1 9 1 7)* showed that fluid which contained col­ loids having osmotic pressure similar to that of plasma-pro- tein could be used as a substitute for blood. He experi­ mented with several materials and found that a 6 per cent solution of gum arable in saline had most of the desired properties. Later, such substances as pectin, cattle plasma, methyl cellulose, polyvinyl alcohol, and ascitic fluids were tested for this use. Some of these materials have antigenic properties, while others cannot be metabolized or excreted, for which reason they tend to accumulate and interfere with the liver function, as reported by Hall (1 9 3 8)> Andersch and

Gibson (193^4-) , and Dick et al (1935) J or apparently lead to atheroma of the arteries as stated by Hueper (19i|2a, 19i|-2b) . Dextran, gelatin and polyvinylpyrrolidine (PVP) are now under intensive study, and are reaching volume production. Amspa- precipitation procedures, a plot was' made of "Per Cent Cumu­ lative Weight Precipitated” versus "Intrinsic Viscosity". The order of the weights of the subfractions is not that of the actual precipitation at a given alcohol concentration but rather that of intrinsic viscosity. It should be no­ ticed that all the weights of the subfractions having al­ most the same intrinsic viscosity were grouped together.

Table 36 gives the integral distribution data for the de­ graded polymer, resulting from the different methods of de­ polymerization. Figure 12 represents the resultant curves

"Integral Distribution Curves".

Differential Distribution Curves. In the determina­ tion of molecular weight distribution, the differential distribution curve is usually the most revealing. This curve results from a plot of "Actual Slopes from the Inte­ gral Curves" versus "intrinsic Viscosity". These slopes represent the rate of change of cumulative weight with change in the intrinsic viscosity . In plotting, slopes were taken off the integral distribution curve at representative intrinsic viscosity intervals and plotted against the-intrinsic viscosity at which they were taken.

Figure' 13 is the resultant curves for the different pro­ cedures of degradation, which indicate that ultrasonic de­ polymerization resulted in a degraded polymer with a very narrow range in molecular weight distribution In contrast TABLE 36

Integral distribution data for the degraded polymers of different procedures of depolymerization

Acid* .. Autolysisw Ultrasound* C u m * l . ^ J'' % Cum»l. Cum* 1. yield wt. g. [ r ( \ yield wt.g. [l] yield wt. g.

0.38 7.5 79.7 0.68 3 7 4 62.7 0.28 3 8 4 72.9 0.29 22.7 72.2 0.58 1 4 25.3 0.275 4 . 6 34*5 0.21 7.1 14-9.5 0.50 4*0 23.9 0.27 12.2 19.9 102 0.18 3.3 i £ 4 0.39 10.8 19.9 0.23 1.8 7.7 0.16 17.0 39.1 0.35 3 4 9.1 0.20 1.7 5.9 0.13 34.0 22.1 0.25 3*1 5.7 0.18 3.2 4 2 0.10 2.9 8.1 0.20 0.8 2.6 0.15 1.0 1.0 0.07 3.9 5.2 0.17 1.0 1.8 o.olj. 1.3 1.3 0.10 0.8 0.8

Acid hydrolysis, autolysis degradation and ultrasound, depolymerization.

-:k ;- Qi0-bal yield of subfractions with almost the same value.

Cumulative weight in grams. — •— M/fra sonic

— o — A uto/ysis

020 0.50 0.40 070 w Jigare/Z. Compari son of integral distribution curves for sub-fractions of degraded dextran 3-Stz resulting from different methods of depo/ynzerzatio/t N 9 /X 0 ,_ \ or 105 to acid and autolysis degradation, which, in turn, lead to broad distributions of molecular weights. 106

DISCUSSION

Factors Affecting Dextran Fermentation

The data presented herein indicate the marked effect of various factors on the production of dextran. The nutrition­ al requirements for the different strains under investigation should be satisfied and maintained, if an increase of activ­ ity is desired. Many investigators have shown that certain amino acids, vitamins and minerals are essential for the

growth of these cultures. -It seems that medium No. 2 , which contains acid hydrolyzed casein, magnesium sulfate and sodi­ um chloride, will provide the desired nutritional require­ ments for growth and production of dextran. The decrease of viscosity of dextran during extended incubation of the culture media appears to be caused mainly by autolysis which merely produces a random decrease in mo­ lecular size. It seems evident that the increase in acidity which coincides with the decrease in viscosity will enhance the liberation of depoiymerizing enzymes after the death of the Leuconostoc cells. The unfavorable effect of mechanical shaking on dextran might be explained on the basis of the mechanism of dextran

synthesis. Hehre (1 9b!) stated that this synthesis involves the repetitive transfer of a glucosyl group from a terminal position In the dextran molecule to a terminal position of 107 a growing dextran molecule. Tills remarkable process seems to be more favored under stationary conditions than under mechanical shaking. The increase in the yield of dextran through early neutralization may be interpreted on the op­ timum pH for the production and the activity of dextransu- crase. Koepsell and Tsuchiya (1 9 5 2) had established that the optimal pH for the production of the enzyme dextransu- crase ranges from 6 .'5-7.0 in the growing culture and that pH 5 .0 -5 . 2 is more favorable for the dextran synthesis. It seems that periodic neutralization to keep the pH around 6 .5 during the initial dextran fermentation will prolong the period which favors the enzyme production to 2l± hours. Such increase in enzyme productivity results In higher yield of the synthesized dextran especially when the pH dropped to 5.0-5.2 and reached I4..2 after 50 hours, in contrast to the regular fermentation where the pH dropped to 6 .2 within 6 hours and then to 3*9 after 2I4. hours, in which case there was not enough enzyme production and decreased activity for dextran synthesis.

Clarification of Dextran

It has long been recognized in the literature that the best procedure for dextran recovery from the culture media is to decrease the viscosity by the addition of 30 per cent alcohol by volume, followed by supercentrifugation to elimi­ nate the bacterial cells. Dextran was then recovered by

! .1 108 further addition of alcohol to the centrifugal ate# This study revealed that such procedure did not remove

all the bacterial cells from the culture media and that a

relationship between the amount of these cells and the dex­ tran opalescence in aqueous solution is present. It seems that the addition of alcohol to the culture media will cause plasmolysis to the bacterial cells to the extent that they can pass the bacterial filters. When the fermented liquor was diluted with water to decrease its viscosity and passed through bacterial filters, the dextran opalescence was re­ moved without any appreciable loss of dextran due to adsorp­ tion which did occur when super cell filtration was tried.

Depolymerization of. Dextran

The production of the desired molecular weight of dex­ tran for the use as a plasma volume expander can be accom­

plished by using the three different procedures under inves­

tigation, namely, autolysis, acid hydrolysis and ultrasound

depolymerization, followed by fractional precipitation of

the degraded polymer to obtain a homogeneous type of dextran. These different procedures resulted in a quite different yield of the desired fractions. *0ut of the original weight

of dextran, 5 to 10 per cent of the desired molecular weight fraction was recovered through autolysis degradation, while 10 to 25 pep cent was obtained using acid hydrolysis, and lastly 80 to 90 per cent of the desired fraction was present 109 when ultrasonic vibration was used for depolymerization. The ultrasound procedure seems to provide us with a new, efficient and easily controlled method for the process­ ing of dextran suitable for use as a plasma expander. The molecular weight distribution of the degraded poly­ mer, resulting from these different procedures showed that autolysis and acid hydrolysis lead to a polydispersed poly­ mer with a wide range of molecular size, while ultrasound produced a polymer with a narrow band of molecular weights. It would appear from these results that there Is an es­ sential difference In the mechanism of these methods of de­ polymerization. In the case of autolysis and acid hydroly­ sis, the degradation seems to be a random cleavage of the glycosidic linkages in the molecules to form a reducing oligosaccharides in contrast with ultrasound, which frac­ tures the dextran molecules to produce a non-reducing oligo­ saccharide. This fracturing may Involve carbon-carbon bonds with a preferential degradation of higher molecular weight material. This fragmentation, resulting in a non-reducing oligosaccharides with somewhat different flexibility and configuration than the other reducing oligosaccharides, may explain the observation of the relation between the molecu­ lar weight as measured by the light-scattering and the square of the intrinsic viscosity. It was found that the molecular weight values of these different fractions from acid hydroly­ sis and autolysis followed the equation: 110

M = l.i+2 x 10^ x [ ^ 2

The ultrasonic fractions and subfractions yielded mo­ lecular weights which were larger for a given intrinsic vis­ cosity than provided for by the above equation. This may throw some further light on the mechanism of the degradation as previously stated. The starting material before degrada­ tion is in all likelihood made up of a large rod-like molec­ ular ’'coils'1 of relatively large cross-sections. "Within these molecules are the dextran chains which are folded in a compact arrangement. It is believed that acid hydrolysis and "autolysis” cause these chains first to unfold and then to break along the glycosidic linkages, while ultrasound does not unfold the chains but fractures the bundle of chains at bonds other than the glucosidic linkages.

Physico-chemical Properties A modified procedure for fractional precipitation was accomplished using partial precipitation with isopropyl al­ cohol and low temperature decantation. This procedure proved to be very efficient and economical from the standpoint of using less alcohol, it avoids the two liquids which appeared when aqueous-alcohol solvent contained 30— per cent alco­ hol and provides us with more homogeneous type fractions. The priniciple of this method is based on three main obser­ vations which were made during the fractionation process: 3 cher (1952), stated that at the present time, dextran shows the greatest promise of meeting the requirements of a good plasma expander. Dextran is a glucose polymer formed as a viscous gel during the growth of certain strains of Leuconostoc mesen- teroides and other soil Streptococci. Sucrose was found to be the only suitable carbohydrate substrate. The major problems in the production of clinical dextran for use as a blood expander are:

A. Clarification of the aqueous solution An aqueous solution of dextran has a faint opalescence, which gives it a blue tinge. This opalescence gives an un­ desirable appearance and indicates a high nitrogen content, which might play an important role in the antigenic proper­ ties of dextran. As will be seen later in the text, the bacterial cells of Leuconostoc mesenteroides are probably the main cause of this opalescence because it was found that when the bacterial cells are removed, the dextran solution becomes clear.

B. Controlling the polymer molecular size The usefulness of dextran as a plasma expander is highly

'V dependent upon the size of its molecule. G-ronwall and Ingel-

man (19i]-5 ) reported that the molecular weight of the native dextran is in the order of many millions. Its use in this Ill

(1 ) Low concentration of dextran. Decrease in concen­ tration allows better means of controlling the fractionation procedure and lessens the mechanical contamination by the small size molecules during the fractionation of the high molecular weight polymers.

(2 ) The fractionation can be made at low temperature

1 0 - 1 2 C which will cause most of the fractions to be separat­ ed at lower alcohol concentrations than required at 25 G.

(3 ) When alcohol is added to the aqueous solution of dextran, the dextran molecules start to precipitate, not in the form of a solid particle but in a form of viscous mater­ ial which precipitates to the bottom of the container. It seems that this separated layer contained less alcohol than the surrounding media, accordingly if the temperature is lowered to 0 - 2 C, this separated fraction will be partially frozen much earlier than the surrounding aqueous-alcohol solvent which contains the unprecipitated fractions, and thus can be recovered easily after the decantation of the aqueous- alcohol solvent. A relation between the intrinsic viscosity of the fair­ ly homogeneous fractions and its weight-average molecular weight as measured by light-scattering was found to follow a straight line function over a range of 20,000 to 180,000. The slope of this straight line was found to be:

M = 1.14-2 x 1 0 ^ All the molecular weight of the fractions recovered 112 from different procedures of degradation followed this re­ lationship except those of ultrasound which suggests that a fundamental difference in the flexibility and configura­ tion of the dextran molecules after irradiation wTith ultra sound, waves. 113

SUMMARY

(1 ) A modified procedure for fractional precipitation of dextran, to overcome the two liquid phases, was established by using a partial precipitation and low temperature decan- tation procedure.

(2 ) Isopropyl alcohol was found to be the best agent for the dextran precipitation, and that a 60 per cent by volume of that alcohol was the most effective concentration for the dextran recovery.

(3 ) Dextran is hygroscopic and thus its concentrations in aqueous solution should be made by refractive index mea­ surements, especially when the concentration of the polymer must be determined accurately. (Ip) Measurements of dextran concentrations can be ac­ complished fairly well by using the refractive index mea­ surements.

(5 ) Some physico-chemical experiments were made on the dextran, its fractions and subfractions. (6) Twenty different samples of fairly homogeneous type dextran resulting from three different procedures of degradation was used for molecular weight measurements. Us­ ing light-scattering measurements, the relation between the intrinsic viscosity of these samples and their weight-aver­ age molecular weight were found to be a straight line func- lilt- tion in the range between 20,000 and 1 8 0 ,0 0 0 : /■

M = 1.4.2 x 10^ x

(7) All the weight-average molecular weight values of these different samples followed this relationship except those of ultrasonically depolymerized dextran which fall just a little above the curve. This may be due to a funda­ mental difference in the flexibility and configuration of the dextran molecule. (8 ) Certain factors affecting the dextran fermenta­ tion and its production, were studied, using different strains on comparative media. (9) The relation between pH, total acids, dextran yields and its molecular weight as measured by the relative viscosity of 6 per cent aqueous solution, during the dex­ tran fermentation were reported. (10) Production of low molecular weight dextran through autolysis, resulted in a fairly high polydispersed polymer, in respect to its molecular weight. (11) Autolyzed dextran may be due to the liberation of an autolyzed enzyme after the death of the bacterial cell, which seems to coincide with the increase of total acids, during the course of dextran fermentation. (12) Mechanical shaking is unfavorable for the dextrai production by the three different strains under investiga­ tion. 115 (13) Periodic neutralization of acidity early in the dextran fermentation resulted in a high yield of high molec­ ular weight dextran. (II4.) Dextran opalescence in aqueous solution is due to bacterial cells and their protoplasmic substances. Elimina­ tion of this opalescence was accomplished by bacterial fil­ tration of the fermented media after it had been diluted with 20 per cent distilled water. (15) The different procedures for the degradation of dextran, namely, acid, autolysis and ultrasonic, were de­ scribed. (16) Certain factors affecting the degradation by each of these procedures have been studied. (17) A new procedure for the depolymerization of dextran using ultrasonic vibration was accomplished. (18) The molecular weight distributions for the degraded polymer resulting from each procedure were reported. Acid and autolysis degradation resulted in a wide range in molec­ ular size, in contrast to ultrasonic which gives a fairly narrow range. (19) Compa,rison of the three different methods of degra­ dation for the production of dextran with physico-chemical properties of a plasma extender, showed that acid and autoly­ sis degradation will result in a low yield of the desired fraction, while ultrasonic depolymerization will give a very high yield, as high as 90 per cent of the original polymer 116 will be, in th.e range of tiie desired molecular size. 117

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Fitzgerald, J. G. 1933 0n the nature of antigens. Trans. Roy. Soc. Canad., Third Series, 27, Sect. V, 1-9. Fowler, P. L., Buckland, I. E . , Brauns, P., and Hibbert, H. 1937 Studies on reactions relating to carbohydrates and polysaccharides LIII. Structure of the dextran synthesized by the action of Leuconostoc mesenter- oides on sucrose. Can. J. Res'.", B 15> I4.8 6 -I4.57 • Gaines, S., and Stahly, G. L. 19l{-3 The growth requirements of Leuconostoc mesenteroides and preliminary studies on its use as an assay agent for several members of the 119 vitamin B complex. J. Bact., 1+6, i+1+1-1+1+9. Gardner, E. Jr. 1952 Factors effecting the production of dextran "by Leuconostoc mesenteroides. Master1s Thesis, The Ohio State University, Columbus, Ohio.

Georges, L. ¥., Miller, I. L., and Wolfrom, M. L. 191+7 Crystalline octaacetate of 6- -D-glucopyronosido-B- D-glucose. J. A. Chem. Soc., 69, 1+73. Goldenberg, M. , Crane, R. D . , and Popper, H. 191+-7 Effect of intravenous administration of dextran, a raacromo- lecular carbohydrate, in animals, Am. J. Clin. Path. 17, 939-91+8. Gronwall, A., and Ingelman, B. 191+5 Dextran as a substi­ tute for plasma. Nature, 155, 1+5- Gronwall, A. , and Ingelman, B. 191+8 Manufacture of infu­ sion and injection fluids. U.S.Pat. 2,1+37,518.

Hall, W. K. 1938 The effects of intravenous injections of acacia upon certain functions of the liver. Am. J. Physiol., 123, 88. Hartman, F. W. 1951 Tissue changes following the use of plasma substitutes. A.M.A. Arch. Surg., 6 3 , 728-738. Hassid, W. Z. , and Barker, H. A. 191+0 The structure of dextran synthesized from sucrose by Betacoccus arabi- nosaceus (orla Jenson). J. Biol. Chem. 131+, 163-170. Hehre, E. J. 191+1 Production from sucrose of a serologic­ ally reactive polysaccharide by a sterile bacterial ex­ tract. Science, 93, 237-238. Hehre, E. J. 191+3 Comparison of dextran synthesis by Leu­ conostoc enzyme with starch synthesis by potato phos- phorylase. Proc. Soc. Exptl. Biol. Med., 51+, 21+0-21+1.

Hehre, E. J. 191+6 Studies on the enzymatic synthesis of dextran from- sucrose. J. Biol. Chera. , 163, 221-232.

Hehre,, E. J. 1951 Enzymic snythesis of polysaccharides: a biological type of polymerization. Advances in Enzymol., 11, 291+-337* Hehre, E. J., and Sery, T. W. 1952 Dextran-splitting an­ aerobic bacteria, from the human intestine. J. Bact., 6 3 , 1+21+-1+2 6 . 120 Hehre, E. J., and Sugg, J. Y. 191+2 Serologically reactive polysaccharides produced through the action of bac­ terial enzymes. I Dextran of Leuconostoc mesenter- oides from sucrose. J. Exp. Med., 75, 339-353.

Hogan, J. J. 1915 The intravenous use of colloidal (gelatin) solutions in shock. J.A.M.A., 6^, 721.

Hucker, G-. J., and Pederson, C. S. 1930 Studies on the Coccaceae, XVI. The genus Leuconostoc. N.Y. Agric. Exp. Sta. Tech. Bui., 167, 1-tJo.

Hueper, W. C. 19l+2a Experimental studies in cardiovascular pathology: pectin atheromatosis and thesaurosis in rab­ bits and in dogs. Arch. Path., 3l+* 883-901. Hueper, W . G. 19l+2b Experimental studies in cardiovascular pathology: Effect of intravenous injections of solu­ tions of gum arable, egg albumin and gelatin upon the blood and organs of dogs and rabbits. Am. J. path., 1 8 , 895-

Ingelman, B. 191+7 Dextran and its use as a plasma substi­ tute. Acta Chem. Scand., 1, 731-738.

Ingelman, B. 191+8 Enzymatic breakdown of dextran. Acta Chem. Scand., 2, 803-812.

Ingelman, B., and Hailing, M. S. 191+9 Some physico-chemi­ cal experiments of fractions of dextran. Arkiv Kemi, 1 , 61-80.

Ingelman, B. and Siegbahn, K. 191+1+ Dextran and Levan mole­ cules studied with the electron microscope. Nature, 151+, 237.

Jeanes, A., Haynes, C. ¥., Wilham, C. A., Rankin, J. C., and Rist, C. E. 1952 Chemical characterization and classi­ fication of dextran from one hundred strains of bacteria. Abstracts of papers, 122nd National A.C.S. meeting, September ll+-19th, 1952.

Jeanes, A., Wilham, C. A., and Miers, J. C. 191+8 Prepara­ tion and characterization of dextran from Leuconostoc mesenteroides. J. Biol. Chem., 178, 603-615* Jeanes, A., and Wilham, C. A. 1950 Periodate oxidation of dextran. J. Am. Chem. Soc., 72, 2655-2657* form will cause injurious reactions. However, dextran depo­ lymerized uniformly to a molecular size of the same order as that of plasma protein, is quite harmless.

C. Molecular weight homogeneity Dextran and its partially degraded products are polydis- persed, i.e., consists of molecules of the same chemical com­ position but with different molecular weights. Its use in this form is very dangerous. The high molecular weight poly­ mer will cause injurious effects to the body tissues, while the low molecular weight material will literally leak through the capillary walls and be rapidly excreted from the kidneys. As will be seen later in the text, a promising new procedure for the degradation of dextran was accomplished. This pro­ cedure involves the use of ultrasonic vibration for the de­ polymerization of the high molecular weight polysaccharide to a lower molecular weight polymer with a fairly high per-

V centage of homogeneity in respect to its molecular size, as contrasted to the acid and autolysis hydrolysis methods which resulted into a highly polydispersed material.

STATEMENT OP THE PROBLEM This dissertation is concerned with the study of factors affecting the production of dextran having physico-chemical properties of a plasma volume expander. The purpose of this investigation was threefold: 121

Kabat, E., and Berg, D. 1952 Production of precipitins and cutaneous sensitivity in man by injection of small amounts of dextran. Ann. N.Y. Acad. Sci., 55, I4.7i-J4.75.

Koepsell, H. J., and Tsuchiya, H. M. 1952 Enzymatic syn­ thesis of dextran. J. Bact., 6 3 , 293-295. Eevi, I., Hawkins, V/. L. , and. Hibbert, H. 19J4-2 Studies on reactions relating to carbohydrate and polysaccharides. LXVI. Structure of dextran synthesized by the action of Leuconostoc mesenteroides on sucrose. J. Amer. Cheiru Soc., SZj!, 1 9 5 9 - 1 9 6 6 . Liesenberg, and Zopf, W. 1892 Ueber den sogenannten Fro- schlaichpilz der eiiropaischen Rubenzucker und der ja- vanischen Rohrzuckerfabriken. Gent. Bakt., 12, 659- 66l •

Lockwood, A. R., James, A. E., and Pautard, P. G-. 1951 Studies on the breakdown product of dextran formed by ultrasonic vibration. Research, 14., 14.6 .

Morhead, P. P. 1950 Ultrasonic disintegration of cellulose fibers before and after acid hydrolysis. Textile Res. J • , 2 0 , 5i|9-553. Niven, C. P. Jr., Smiley, K. L., and Sherman, J. M. I9J4-I The polysaccharides synthesized by Streptococcus sali- varius and Streptococcus bovis. J. Biol. Chem., II4.O, lu5-lo9. Pulaski, E. J. 1952 Clinical status of plasma substitutes. Chem. and Eng. News, A. Chem. Soc., 30, 2187-2189.

Prudhomme, R. 0., and Graber, P. 19^-9 (Ins. Pasteur, Paris) The chemical action of ultrasonic on certain aqueous solutions. J. Chem. Phys., I4.6 , 323-331.

Renfrew, A. G., and Cretcher, L. H. 1914-9 Partially hydro­ lyzed dextran. J. Amer. Pharm. Assoc., 3 8 , 177-179.

Snell, E. E. , Strong, p. M., and Peterson, IV. H. 1938 Pan­ tothenic and nicotinic acids as growth factors for lac­ tic acid bacteria. J. Am. Chem. Soc., 60, 2825.

Sobue, H., and Hawai, H. 19J+U- Depolymerization of chain hagb- molecular compounds. Chem. High Polymer (Japan), 1, 65-70. Stacey, M. 19^.2 Enzymatic production of bacterial polysac­ charides. Nature, 114-9, 639. 122 Stacey, M. 195l Degradation or dextran by ultrasonic waves. Research, I4 ., I4.8 .

Stacey, M. and Pautard, F. G. 1952 Thermal degradation of dextran. Chem. & Ind;, 1058-1059. Stacey, M. , and Youd, F. R. 1938 CCL A note on the dex­ tran produced from sucrose by Betacoccus arabinosaceus haemolyticus. Biochem. J., 32“ 194.3 -I9i4.5~

Stahly, G-. L. 191+3 Method of making dextran. U.S. Pat. 2,310,263. Szent-Gyrogi, A. 1933 Chemical and biological effect of ul­ trasonic radiation. Nature, 131, 278. Sugg, J. Y., and Hehre, J. . E. 191-1-2 Reactions of dextrans of Leuconostoc mesenteroides with the antiserums of leuconostoc and of types 2 , 20 and 12 pneumococcus. J. Immun. , 4_3, 119-128. Swanson, M. A., and Cori, C. F. 191-1-8 Studies on the struc­ ture of polysaccharides. J. Biol. Chem., 172, 797-811}.. Tarr, H. L. A. , and Hibbert, H. 1931 Studies on reactions relating to carbohydrates and polysaccharides. XXXVII. The formation of dextran by Leuconostoc mesenteroides. Canad. J. Research, I4-Il4.-I4.2 7 • Tsuchiya, H. M. , Jeanes, A., Bricker, H. M.> and Wilham, C. A. 1952 Dextran-degrading enzyme from molds. J. Bact., 61+, 513-519. Tsuchiya, H. M., Koepsell, H. J., Corman, J., Bryant, G . , Bogard, M. 0., Ferger, V. H., and Jackson, R. W. 1952 The effect of certain cultural factors on production of dextransucrase by Leuconostoc mesenteroides. J. Bact., 6I4., 521—526.

Van Tieghem, P. 1878 Sur la gomme de sucrerie. Ann. sci. nat. Botan. et biol. vegetale, 7* 180-203.

Weissberger, A. 191-1-9 Technique of organic chemistry. I Physical methods of organic chemistry. Interscience Publisher, Inc., New York. Weissler, A. 1950 Depolymerization by ultrasonic irradia­ tion; the role of cavitation. J. Appl. Phys., 21, 171-173. 123 Weissberg, S. G-., and Isbell, H. S. 1952 Molecular prop­ erties of plasma substitutes. National Bureau of Stan­ dards Report 1713, June 13. YJeissler, A., Cooper, H. YJ., and Snyder, S* 1950 Chemical effect of ultrasonic waves; oxidation of potassium io­ dide solution by carbon tetrachloride. Am. Chem. Soc. J., 72, 1769-1775. Yihiteside-Carlson, V., and Carlson, W. Yi. 19i{-9a The vita­ min requirement of Leuconostoc for dextran synthesis. J. Bact., 58, 135-114-1. Yfhiteside-Carlson, V., and Carlson, W . YJ. 19l)-9b Studies of the effect of para-aminobenzoic acid, folic acid, and sulfanilamide on dextran synthesis by Leuconostoc. J. Bact., 58, llj.3-114-9. YJhiteside-Carlson, V., and Carlson, W. W. 1952 Enzymatic hydrolysis of dextran. Science, 115, l-i-3. YJhiteside-Carlson, V., and Rosano, C. 1951 The nutritional requirements of Leuconostoc dextranicum for growth and dextran synthesis. J. Bact.^ 62, 583-589. YTise, B., and Ensminger, D. 1952 Putting ultrasonic to work. Product Engineering, 38, 180-185. V7ood, R. YJ., and Loomis, A. L. 1927 The chemical effects of high frequency sound waves. I. A preliminary sur­ vey. J. Am. Chem. Soc., I4.9 , 3086. Zettnow, E. 1907 Uber Froschlaichbildungen in Saccharose enthaltenden Plussigkeiten Zeitschr. fur Hygiene, 57, 1514--173. Zozaya, J. 1932a Carbohydrates absorbed on colloids as antigens. J. Exp. Med., 55, 325-351. Zozaya, J. 1932b Immunological reaction between dextran polysaccharide and some bacterial antisera. J. Exp. Med., 55, 353-360. 121+

AUTOBIOGRAPHY

I, Mostafa Kamal Hamdy, was born in Cairo, Egypt, May 27» 1921. I received my primary and secondary school edu­ cation in the public schools of Cairo, Egypt. My under­ graduate training was taken at Fouad I University, College of Agriculture, from which I received the Bachelor of Science degree in 191+1+. From 191+1+ to 191+7j I held the position of

instructor in the Botany Department, College of Agriculture,

Alexandria University, Alexandria, Egypt. In 191+7, I held the same position in the Bacteriology Department, College of Agriculture, Fouad I University, during which time I re­ ceived the degree Master of Science in 191+9, from the same

University. In 1949 I was appointed assistant lecturer in the Bacteriology Department. In March, 1950 I came to The

Ohio State University on leave of absence to complete the requirements for the degree of Doctor of Philosophy. (1) To study the factors which affected the production of dextran during the fermentation procedure.

(2) To clarify the opalescence of its aqueous solution. (3) To contribute information on the production of clinical dextran with different procedures of degradation, namely, acid hydrolysis, autolytic enzymes and ultrasonic vibration. 6

LITERATURE REVIEW

Production of Dextran Dextran has been known since the nineteenth century. It was sometimes encountered as a slime in large globular * masses during the processing of cane and beet sugar, where it increased the viscosity of the sugar solution and re­ tarded filtration and crystallization. When these globular masses were examined microscopically, they were found to be made up largely of chains of encapsulated cells. While•investigating an outbreak of slime in a sugar fac­ tory, Cienkowski (1878) isolated and studied the organism responsible, to which he gave the name Ascococcos mesenter­ oides as it was similar to the Ascococcos which had been pre- - viously described by Billroth (l87i-{-). Van Tiegham (1878), who studied the same organism, decided that it was different from that of Billroth's, and gave it the generic name Leuco­ nostoc . Hucker and Pederson (1930) reported that this de­ scriptive name was given to these organisms because of the resemblance of their slimy masses to those of the blue-green

alga known as Nostoo. Liesenberg and Zopf (1892) and Zettnow

(1907) described methods of isolation. The latter stated that the optimum temperature for growth was 30-32 C. Dextran is produced by the action on sucrose of certain strains of chain-forming cocci, classified by Hucker and Ped­ erson (1930), as the Family Coccaceae, Tribe Streptococceae, 7 Genus Leuconostoc, Species L. mesenteroides and L. dextrani- cum. These investigators found that species of Leuconostoc which ferment sucrose, produce in addition to dextran, approx­ imately 30 per cent L-lactic acid, 5 per cent acetic acid, 10 per cent D-mannitol.

Niven et al (191+1) reported that a few strains of Strep­ tococcus salivarius and Streptococcus bovls are also able to synthesize, from sucrose and raffinose, an insoluble carbo­ hydrate which seems to be a dextran. Optimum conditions for the preparation of dextran have been determined by Tarr and Hibbert (1931) y who stated that sucrose was the only suitable carbohydrate substrate for the production of dextran. They also reported that it is neces­ sary to activate the culture by several passages of the or­ ganism through suitable media before dextran is formed. This was found to be important in the present investigation. Many investigators, using chemically defined media, have shown that certain accessory substances are necessary for the growth of Leuconostoc. Snell et al (1938) reported that L. mesenteroides required pantothenic acid, while Bohonos et al (191+1 > 191+2) observed that added pyrodoxine was stimulatory. Gaines and Stahly (191+3) found that thiamin, calcium panto­ thenate and nicotinic acid were essential for L. mesenter­ oides 535. Whiteside-Garlson and Carlson (19l+9a, 19l+9b) es­ tablished that thiamin, nicotinic acid and pantothenic acid were important for strains 535> 583 and L. dextranicum 8086 8 (ATCC) but that biotin appeared to be non-essential in su­ crose media. They also found that p-aminobenzoic acid is not involved in dextran synthesis by these strains. The nutri­ tional requirements of L^_ dextranicum, strain "elai" were re­ ported by White-Carlson and Rosano (1951).

The following substances have been shown to increase the yield of dextran: molasses (Carruthers and Cooper 1936), commercial maple syrup (Stacey and Youd 1938) s raw fig (Bouil' lenne 1938), yeast extract (Baker and Stacey 1939), raw cane sugar (Carlson and Stahly 1939), water extracts of waste sug­ ar-refining charcoal (Stahly 191+3) » sterile Staphylococcus aureus filtrates (Carlson 1939), magnesium and ammonium sul­ fate (Hassid and Barker 191+0), and a mixed culture with Sac- charomyces_cerevisiae (Stacey 191+2). The enzyme synthesis of dextran and its mode of action has been described by Hehre (191+1, 191+6, 1951) and Hehre and Sugg (191+2). Rapid formation and high yield of the enzyme^ was reported by Koepsell and Tsuchiya (1952). Certain cul­ ture factors affecting dextransucrase production have been studied by Tsuchiya et al (1952). The role of phosphoryla­ tion was investigated by Hehre (191+3) who established that . dextran synthesis from sucrose by Leuconostoc enzyme does not require t£e inediatipn of phosphorylated sugar.

The Structure of Dextran Studies on the chemical structure of dextran were made by Fowler et al (1937) and Hassid and Barker (19^0), and their results were confirmed by Levi et al (191+2). The following chemical formula has been suggested by these in­ vestigators:

a 1: 6ft. 1: 6r„ 1: 6^1: 6Pt1

or

Hassid and Barker (191+0) stated that the positive ro­ tation and the downward mutarotation during the hydrolysis would indicate that the 1,6 glucosidic linkage has the ex: - configuration. It follows that dextran consists of long main chains with side chains, in which the predominant glucopyranosidic linkage Is^-1,6 linkage, while -1, linkage occur at the points of branching. Jeanes and Wilham (1950) and Jeanes et al (1952) reported that the non-1,6 linkages were all **-1,1+ linkage or a mixutre of these with 1,3 linkage. Electron microscope studies have been reported by In­ gelman and Siegbahn (191+1+) and Ingelman and Hailing (191+9), and they have interpreted the micrographs as providing a confirmation of the structure given above.

Immunological Properties of Dextran Zozaya (1932) reported that nitrogen free dextran was not antigenic but could be rendered antigenic by adsorption 10 upon a colloidal carrier* such, as colloidin. He also found

that dextran reacted immunologically -with antisera of pneu­ mococci, certain of the Salmonella group and some strains of Streptococcus viridans. Sugg and Hehre (1914-2) observed that dextran cross-reacted with types 2, 12 and 20 antipneu- mococcal sera. Fitzgerald (1933) found that the antibody response of rabbits toward 1.5 per cent aqueous dextran solu­ tion varied with the nitrogen (i.e., bacterial) content of dextran.and that no antibodies were produced when the nitro­ gen content of dextran was below 0.2 per cent, Kabat and Berg (1952) established that native dextran and a high molec­ ular weight fraction from a sample of clinical Swedish dex­

tran were antigenic. Amspacher and Gray (1952) have reported that allergic type reactions occur when dextran is administered to healthy volunteers. Unfavorable clinical reactions to American dex­ tran are far less frequent than that to Swedish dextran. Many explanations have been suggested for these reactions, but the problem remains unsolved and is currently under in­ tensive study.

Degradation of Dextran Gronwall and Ingelman (1914-5) reported the breakdown of the very high molecular polysaccharide dextran to a substance with lower molecular weight by means of partial acid hydroly­

sis. Georges et al (19^-7) used 2 per cent dextran in 30 per ACKNOWLEDGMENT

I wish to take this opportunity to express my sincere thanks and deep gratitude to my advisers, Dr. Harry H. Wei- ser and Dr. Grant L. Stahly, for their guidance, counsel and encouragement that they have copiously given me, towards the fulfillment of this study. On innumerable occasions, Dr. Quentin Van Winkle has munificently assisted and given me invaluable advice in problems of physical chemistry. To him, I owe an immense debt of endless gratitude. I also desire to show my appreciation to both Dr. Jor- gen M. Birkeland and Dr. Chester I. Randles for their spon­ taneous and constant interests, and for suggestions which they so freely gave.

Many thanks to Dr. Prank VJ. Bope of the College of Pharmacy, who has so generously permitted the use of ultra­ sonic equipment. Acknowledgment is also due to Mr. Norman D. Jaynes, technical assistant of the Engineering Experimental station, and Professor Howard D. Smith of the School of Architecture for their help in drawing the figures. I can only express my profoundest thanks to them for their kindness. A multitude of thanks to Miss Jane Kintner, librarian of the Pharmacy and Bacteriology Library, and to all of those who have made it possible for me to conclude this problem.

a o c i o s 11 cent hydrochloric acid at room temperature. Partial acid hy­ drolysis by autoclaving solutions of dextran was described by Renfrew and Cretcher (191+9). They also established that sterilization by autoclaving for thirty minutes caused no measurable change in relative viscosities of 5 per cent neu­ tral solutions of partially hydrolyzed dextrans. Swanson and Cori (19l|-8) established that the **-1,6 linkage is more resistant to acid hydrolysis than the I,!}, linkage. They also reported that the enzymes phosphorylases and the common amy­ lases do not act on dextran. Colin and Belval (I9I4-O) showed that the pancreatin has no effect on dextran, and that under prolonged acid hydrolysis dextran is converted to glucose. Enzymatic degradation has been reported by Ingelman (19l}-8), using extracts from cultures of bacterium Cellvibrio fulva. Jeanes et al (19^8) stated that the low molecular weight dextran, which can be obtained on extended incubation, is due to autolysis. Whiteside-Carlson and Carlson (1952) used a culture filtrate from an Aspergillus sp. to degrade

the high molecular weight dextran.- Tsuchiya et al (1952) reported that 20 strains of Peni- cillium lilacinum, P. funiculosum, P. verruculosum and Spi- caria vlolacea produced potent extra-cellular dextran-degrad- ing enzymes. They also reported that an amylase concentrate of Aspergillus niger, strain N.R.R.L. 330* was capable of degrading dextrans. Stacey and Pautard (1952) studied thermal cleavage under reducing conditions at neutral pH. Lockwood et al (1951) and Stacey (1951) found that degradation can be accomplished under sensitive control using ultrasonic vibration.

Fate of Infused Dextran t Ingelman (1947) and Goldenberg et al (1947-) reported that after repeated large injections of dextran, no accumu­ lation can be detected in the organs and that only a small fraction is excreted in the urine as macromolecular or other carbohydrate, suggesting that most of it is broken down and assimilated by the body. Hartman (1951) reported that dex­ tran is eliminated from the body with relative rapidity and that- with intravenous injections, varying amounts were re­ tained in the liver and the reticulo-endothelial cells. Engstrand and i^berg (1950) reported that dextran is broken down on incubation with feces, Hehre and Sery (1951) have confirmed this observation and have identified the ac­ tive agent as an enzyme elaborated by a gram negative anaer­ obe of the Bacteroides group. Using dextran labelled with Amspacher and Gray (1952) established-that the nonexcreted dextran in animals is converted to glucose, and they could demonstrate radio­ activity in the carcass carbohydrate, fat and protein three days after injection. -13- Physico-chemical Measurements Physico-chemical experiments on dextran fractions have been reported by many investigators to establish the molec­ ular properties which are of importance in medical applica­ tions. Ingelman and Hailing (19l|-9) used viscosity measure­ ments, ultracentrifugation, diffusion and adsorption experi­ ments to characterize some dextran fractions with respect to their molecular weights. They established the following linear relation between the intrinsic viscosity [>^3 and the molecular weight (M) for some partially hydrolyzed dextran fractions in the interval 14-0,000 M ( 3 0 0 ,0 0 0 :

1*0 - 8.2 x 10~7 M / 0.18

Wales, Marshall and Weissberg, as signified in the Bu­ reau Standard Report (1952), studying the intrinsic viscos­ ity-molecular weight relationship for acid hydrolyzed dex­ tran B—5>12, reported using the sedimentation equilibrium method In order to obtain a measure of the polydispersity of the fractions, as well as a weight - average molecular' weight. They established the following relation between the intrinsic viscosity and the weight-average molecular weight of some dextran fractions in the range 20,000 ^ M < 2^0,000: I k

MATERIALS AND METHODS /

Bacteriological

Cultures. Three strains of Leuconostoc mesenteroides were employed throughout the investigation. They were Leu-

conostoc mesenteroides 683“, L. mesenteroides N.R.R.L. B-512 and L. dextranioum " elai,r“~'i’. The cultures were kept in the ice box (6 C). Transfers were carried out each three weeks. For dextran production, the cultures were activated by sev­ eral passages through the same media used for the fermenta­ tion procedure, allowing twelve hours between each inocula­ tion. Media. For the stock culture, the medium used had the following composition per liter: sucrose i+0.0 g yeast extract 1.0 g acid hydrolyzed ^ ^ casein"'*”35' 5.0 g K2HF0j, 5.0 g NaCl 2.0 g MgSOj. 0.022g agar 15.0 g For dextran production, two media having the following ingre* dients per liter were used.

These strains were received from Dr. Carl S. Pederson, New York State Agricultural Experiment Station, Geneva, N.Y. ':h:' This strain was received from the University of Califor­ nia . *:hhc- hydrolyzed casein was obtained from the S.M.A. Corp. 15 Medium No. 1 Sucrose 150.0 g peptone 2.5 g yeast extract 2.5 g K 2HP0k 5.0 g NaCl ^ 2.5 g water extracts of waste sugar-refining charcoal 2.0 ml Medium No. 2

Sucrose 150.0 g acid hydrolyzed casein 5.0 g yeast extract 1.0 g K 0HPO1, 5.0 g NaCl 2.0 g MgSO^ 0.022 g The pH of all media was adjusted to 7.2

Sterilization, inoculation 1and incubation. Steriliza- tion, effected by autoclaving at 15 lbs for 15 minutes, was followed "by cooling and inoculation; incubations were at

25 C.

Preparation of inoculum. The cultures were prepared by inoculating a loopful of active culture into 10 ml of sterile medium in a test tube. After 2l\. hours incubation, a loopful of this medium was transferred to another 10 ml test tube. Twenty-four hours later, the entire contents of the tube were transferred to 100 cc of the same media in a 250 cc Erlenmeyer flask. After 2J+ hours, the culture was used for inoculation. 16

Chemical'* Determination of pH. pH measurements were made by us­ ing a Fisher titrimeter, which was calibrated against a buf­ fer solution of pH 7 each time the apparatus was used.

Total acids measurements. A 10 ml aliquot of fermented culture media was used for titration against a standard solu­ tion of NaOH, using phenolphthalein as indicator. Knowing the volume of alkali used, the percentage number of milli­ moles of total acids could be calculated for the sample.

Precipitation of dextran. Two methods were employed for the precipitation: (1) At the end of the desired incubation period, the fermented media were diluted to decrease their viscosity using 99 P©r cent isopropyl alcohol so as to give 20 per cent alcohol concentration by volume. In order to maintain maxi­ mum agitation, dilution was carried out in a "Waring blendor, after which repeated supercentrifugations were made. The residue consisted largely of bacterial cells. The superna-f tant was stirred mechanically using the Waring blendor while the alcohol concentration was brought up to 60 per cent by volume. Dextran precipitated as a white gummy mass. The supernatant was decanted and dextran was kneaded to remove

"* In the chemical analyses, duplicate experiments were made and the average results are reported. 17 mother liquor, then dissolved in distilled water, and repre­ cipitated by addition of 99 per cent isopropyl alcohol. The cycle of reprecipitation and washing was repeated several times and the dextran was filtered through a Buchner funnel and dried. (2) The precipitation procedure was done after the clarification of the fermented media, as will be shown later in the text. The fermented liquor was diluted with distilled water and filtered through large bacterial filters (Seitz <.or Berkefeld). Isopropyl alcohol was added to the filtrate to

give 50 per cent by volume, and the mixture was kept in the ice box for 24 hours. The supernatant was then decanted and the concentrated aqueous solution of dextran was added to 99 per cent isopropyl alcohol a drop at a time while the alco- ' hoi was stirred vigorously in a "Waring blendor. By use of this procedure the gummy stage was avoided and the dextran was precipitated as a very fine flocculant powder, which was separated from the alcohol by filtration. Mechanical stir­ ring of the precipitated dextran was repeated in 99 per cent isopropyl alcohol after which the dextran was filtered and dried. This method proved to be efficient in the precipita­ tion of large amounts of dextran.

Effect of various precipitating reagents. Joint experi­ ments were made with Spendlove*"' to determine the most effec- *Spendlove, C. is a graduate student in the Department of Bacteriology of The Ohio State University. 1 8 tive concentration of various precipitating reagents in the dextran precipitation. A lp. 3991+ per cent aqueous solution of neutral unhydrolyzed dextran 683 was used. Aliquot por­

tions of 10 ml were placed in 50 cc Erlenmeyer flasks. Dex­

tran was precipitated using different concentrations by vol­ ume of the following reagents:

100 per cent methyl alcohol, 95 per cent ethyl alcohol, mixture of ethanol and acetic acid in a ratio of 60:^0, 99 per cent isopropyl alcohol, and 100 per cent acetone.

The precipitates were allowed to settle for 214. hours in the ice box (6 C). Before decantation of the superna­ tants, the solution was quickly cooled to 2 C which caused

the precipitated dextran to adhere to the bottom of the glass container. The precipitated dextran was removed and - dried in the oven (I4.5 C) for several days. Cooling was made by immersing three-quarters of the Erlenmeyer flask in a mix­

ture of ethanol and dry ice for I4.-7 minutes. The results shown in t a M e 1 suggest the following conclusions:::

(1) The best reagents for the precipitation of dextran

at lower concentration were isopropyl alcohol, ethyl alcohol

and acetone, respectively. (2) The most effective concentration for the precipi­

tation of dextran was 60 per cent by volume of either 99 per

cent isopropyl alcohol or 100 per cent acetone. TABLE 1

Average per cent recovery of a Ip.399U- per cent .solution of dextran 683 using various concentrations of precipitating reagents

$ 100$ Methyl 95$ Ethyl 60$ Ethanol 99$ Isopropyl Reagent Alcohol Alcohol lp0$ Acetic Acid Alcohol Acetone 1 30 23.9 1**7 2i98 50.2 18 i 9

35 J+8.6 S l.k • 2.86 514-.5 53.9 lpO 52.8 53/0 52.0 5k. 2 57.1 kS 51.7 53.li- 5ip.9 5 8 * 59.8 5o 53.3 514-5 5 7 * 62.2 68.1 55 57.3 57.6 59.8 68.3 75.5 60 62.9 58.5 60.9 75.0 78.2 65 70.6 71.1 71.7 77.9 80.2 70 71.9 7Il. 6 71.7 80.6 82.6 75 77.0 73.7 79.8 78.8 81.9

Control 0.27 0.07 0.18 0.36 0.07

75$ reagent amt. 10 ml H2 O 20 Drying of dextran. Dextran was usually dried in vacuum

over anhydrous calcium chloride at 2 $ C. Sometimes drying was done in a i|.5 C oven for I4.8 hours. The weight of the product (dry basis) was recorded and the percentage yield was calculated on the basis of: (1) The initial weight of sucrose used in the experi­ ment . (2) The glucose available from the sucrose. Hehre and Sugg (191^2) reported that the over-all equation for dextran synthesis may be represented as: n sucrose — (D-glucose - H 20)n / n D-fructose dextran

Effect of humidity on weight of dextran. Dextran is a hygroscopic material that can absorb moisture from the atmos- - phere and give rise to incorrect weighing measurements. This experiment was conducted to calculate the percentage increase in the weight of dextran for different lengths of time. Two different samples of dextran were used. Sample ,TaM

was dried and stored in a closed bottle for two weeks. Sam­ ple "b" was weighed immediately following drying and cooling in a desiccator. The samples were weighed quickly, using a chainomatic balance at room temperature, left on the pan of the balance for a definite period of time and reweighed. The latter step was repeated several times. The results TABLE OP CONTENTS PAC-E INTRODUCTION ...... 1 STATEMENT OP THE PROBLEM ...... I|. LITERATURE REVIEW ...... 6 Production...... 6 Structure of Dextran ...... 8 Immunological Properties ...... 9 Degradation of Dextran ...... 10 Pate of Infused Dextran ...... 12 Physico-chemical Measurements ...... 13 MATERIALS AND METHODS ...... lit- Bacteriological ...... lq. Chemical ...... 16 Depolymerization Methods ...... 23 Autolysis Degradation ...... 23 Acid Hydrolysis ...... 26 Ultrasonic Depolymerization...... 26 Physico-chemical ...... 29 Fractionation ...... 29 Viscosity...... 31 Light Scattering ...... 35 EXPERIMENTAL ...... Factors Influencing Dextran Fermentation ....

Effect of strain and nutrients ...... L|i|. 1If feet of extended inciibation...... i.|.6 E f f e c t of mechanical shaking ...... 55 Effect of neutralization...... 57

-ii- 21

TABLE 2

The effect of humidity on dextran*s weight

Time intervals Percentage increase in weight in minutes Sample "a"* Sample

3 0.12 0.25 6 0.20 0.53 9 0.29 0.75 12 0.38 0.95 15 0 .14.0 1.13 18 1.32

21 __ 1.1+5 2 k — 1.60

27 — — 1.73

sample "a" - a previously dried dextran kept in a closed bottle for two weeks sample nb" - dextran weighed immediately following . drying and cooling in a desiccator not completed 22 were tabulated in table 2, from which the following conclu­ sions may be obtained: (1) Dextran is a hygroscopic material. (2) The percentage increase for sample "a" was 0.1+0 per cent for a period of 15 minutes, while sample "b,r was 1.13 per cent for the same period of time. Prom these re­ sults it can be stated that for procedures where dextran concentration is very important as in the intrinsic vis­ cosity measurements, weighing should be made quickly after drying and cooling, and checked using refractive index mea­ surements.

Measurements of dextran concentration. The extent to which a beam of light is bent or refracted when it passes from one substance into another depends on the relative num­ ber of atoms in the two substances which are in the path of light (i.e., the concentration), and the kind of atoms and their arrangements within the molecules. Accordingly, the refractive index, which is a quantitative measure of the re­ fraction of light, may be used to determine concentration of materials as well as identity and purity of chemical com­ pounds.

Throughout this investigation dextran concentrations in aqueous solution were -checked using a carefully cali­ brated Bausch and Lomb Dipping Refractometer which, as stated by Daniels (191+8), can be used easily under laboratory condi- 23 tions where temperature is carefully controlled. The mea­ surements were made in a constant temperature at 0 C. Concentration was calculated using the following equa­ tion:

where "c11 is the per cent concentration, " n" is the dif­ ference of refraction between the solvent and the dextran solution and "k" is a constant. Determination of "k" was made by the measurements of the refractive index of different concentrations of dex­ tran in aqueous solution and the refractive index of the solvent "distilled water". The results of these experiments are recorded in table 3j from these data "k" was calculated using the previous equation and found to be 0.00176. To check this .value, known concentrations of dextran in aqueous solution were made. The refractive index of the solvent and dextran solutions were measured from which

the percentage concentration "c" was calculated using the constant "k". The results of this work were recorded in

table I4., from which the accuracy of this procedure may be verified.

Depolymerization Methods

Autolysis degradation. Production of degraded dextram through autolysis is based on incubation of the fermented media, extended beyond the time for maximum viscosity, as 211-

TABLE 3

Determination of the "k" constant for the measurement of concentration of dextran in aqueous solution, using the Dipping Refractometer

0 % Refractive index'*' by Solvent Solution n** ir = A n we ight ? nJ> c

0.4317 1.33320 1.333970 0.000770 0.001780

0.6902 1.33320 1.334389 0.001189 0.001723

0.8677 1.33320 1.334740 0.001540 0.001760 0.9456 1.33320 1.334970 0.001770 0.001780

average 0.001760

Refractive index measurements were made at 0 G on aqueous solutions of dextran and distilled water. n = the difference between the refractive index of the dextran solution and the solvent.

i 25

TABLE I4.

Measurement of percentage concentration using the Dipping Refractometer

c % Refractive index 056 b y of calculated*'"' we ight solution

0.80 g. 1 . 33I4.7J4. 0.875 g. 1.60 g. 1.335978 1.570 g.

2.00 g. 1.33667 2.05 g.

Ij-.OO g. 1 . 3 ^ 0 2 1 2 3.99 g.

The concentration was measured using calculated nk" with the refractive index of the solution and the solvent. /

26 reported by Jeanes et al (19I4.8 ). Two 6. liter Erlenmeyer flasks, each containing 5.5 liters of sterile media (No.2) were inoculated with active culture of mesenteroides 683 and B“5l2, as previously described using 5 per cent inocu­ lum. The flasks were incubated for twenty-one days, during which they were shaken at intervals of three days. The fer­ mented liquor was diluted and clarification procedure was carried out and the "autolyzed" dextran was recovered by precipitation using isopropyl alcohol. L. mesenteroides 683 gave a 60 per cent yield and a relative viscosity of 3.3 of a 6 per cent aqueous solution while L. mesenteroides B-512 gave a 66 per cent yield and a relative viscosity of Ip. 7 of a 6 per cent aqueous solution.

Acid hydrolysis. Partial acid hydrolysis of 6 per cent solution of high molecular weight dextran was accomplished using 0.1 N HC1 at 60 C in a water bath. The hydrolysis was followed by measuring the relative viscosity of the product. The degradation was stopped by cooling and neutralization by using 5N NaOH. The aqueous solution was clarified and the partially hydrolyzed dextrans were precipitated.

Ultrasonic depolymerization. Carlin (19ip9) stated that the term "ultrasonic" refers to vibrational waves of a fre­ quency above the range of human hearing which includes all those waves of a frequency of more than 20,000 cycles per second. Wood and Loomis (1927) were the first to report 27 that ultrasonics are able to produce a great diversity of phenomena pertaining to the realm of colloid chemistry. Carlin (1914-9) stated that the most popular types of electromechanical converting systems are the piezoelectric and magnetostrictive. There are several means of producing ultrasonic waves. The most common is the quartz crystal.

In this investigation the piezoelectric sound generator, a

device for transforming high-frequency electric oscillations

into mechanical vibratory energy through the use of the pie-

zoelectric effect , which causes the quartz crystal to al­

ternately expand and contract in the presence of an alter­

nating electric field, was used. Work has been done using ultrasonic vibrations for the depolymerization of compounds. Changes in the viscosity of - colloidal solutions as well as splitting of macromolecules were reported in the literature. Szent-G-yorgi (1933) re­ ported that ultrasonic radiation decomposed cane sugar into mono-saccharide and that highly polymerized compounds are depolymerized by such radiation as Indicated by a rapid fall

in their viscosity. Brohult (1937) stated that the hemocy- anin molecule is split into fragments of one-half and one- eighth its original molecular weight if irradiated with

"if pressure is applied to a crystal system without center of symmetry, certain faces become electrically charged; con­ versely, if a potential difference is applied to certain faces of the crystal, it contracts or expands according to the direction of the applied voltage. These phenomena are called piezoelectric effects.

t 28 ultrasound. He also noticed that this action was accompan - ied by a rise in temperature to about 1+0 C. Sobue and Hawai

(191+6 ) reported that degradation of polystyrene can be accom­ plished in dilute solution and that the longer molecules were depolymerized rapidly and more completely than the shorter ones. Morehead (1950) stated that native and regenerated cellulose fibers are broken into thin "fibrills" through the action of ultrasound.

The mechanism of ultrasonic depolymerization, and dis­ persion is also discussed in the literature. Prudhomme and

Graber (191+9) stated that the rupture of polystyrene mole­ cules is due entirely to cavitation and that the time re­ quired for depolymerization is inversely proportional to the intensity of the waves and proportional to the concen­ tration of the solution. They also reported that polymers of polystyrene of different molecular weight are reduced to the same length of chain. Weissler (1950) confirmed that the depolymerization of polystyrene is due to the cavitation action. He also stated that the molecular weight of poly­ styrene (as measured by intrinsic viscosity) decreased to to about one-tenth of its initial value.

Carlin (191+9) * Weissler et al (1950), and Wise and Ens- minger (1952), explained this cavitation action. They inde­ pendently stated that the energy in ultrasonic waves is car­ ried through the medium by the back and forth motion of the molecules, which produces alternate compressions and rarefac­ 29 tion, that a c t u a l l y disrupt the media. In addition, there are considerable alternations of pressure between compres­ sion and tension at a given point in the liquid. They also stated that one effect of such great changes in pressure is cavitation which is the alternate formation and violent col­ lapse of small bubbles or cavities in the liquid. In this investigation, Model U-300 Ultrason apparatus was used, which produces a maximum of 2jp0 acoustical watts in an oil bath with a small variable frequency range. The Model U-300 Ultrason generator produces a frequency of Lj-5>0,000 cycles per second and a maximum electrical output of 900 watts. During depolymerization a rise in tempera­ ture of the dextran solution was noticed. To avoid such a rise in temperature, a cooling unit was immersed in the con­ tainer in which the depolymerization was made.

Physico-chemical

Fractional precipitation. Degraded dextran consists of molecules of diverse molecular weights. Fractionation experiments using 99 per cent isopropyl alcohol were done to investigate the molecular weight distribution and to ob­ tain homogeneous dextran for physico-chemical studies. The use of a precipitating agent or non-solvent such as isopro­ pyl alcohol to separate polydisperse preparations of dextran into several molecular weight fractions, each with a smaller range of molecular weights than the starting material, is 30 "based upon the fact that the solubility of dextran in water- isopropyl alcohol mixtures is dependent on the molecular weight of the dextran and the isopropyl alcohol concentration.

As in the case of other macro-molecular materials, the solu­ bility of dextran in a given solvent decreases as molecu­ lar weight increases. Also, for dextran of a given molecu­ lar weight, solubility in aqueous solutions decreases as iso- propyl alcohol concentrations increase. Therefore, on the addition of isopropyl alcohol to aqueous solutions of poly­ dispersed dextran, the high molecular weight fractions pre­ cipitate first, and with each succeeding addition of isopro­ pyl alcohol, lower and lower molecular weight fractions are precipitated.

The optimum conditions for fractionation were studied and the following procedure was established for the frac­ tional precipitation, avoiding the two liquid phases which appeared when aqueous-alcohol solvent contained 30”^-3 P®^ cent of alcohol at room temperature.

(1) An aqueous solution of low concentration of dex­ tran (2-3 per cent) was used. It was found that the lox^er the concentration, the more accurate the fraction procedure.

(2) Under mechanical stirring the desired amount of alcohol was added slowly while the temperature was kept at 10-12 C, using alcohol and ice bath.

(3) The temperature of the solution was raised to 2f>-30 C to redissolve the precipitated fraction, cooled to TABLE OP CONTENTS (Continued) Page Clarification of Dextran...... 62 Dextran opalescence ...... 62 Methods of clarification of dextran ...... 63 A. Use of super-cell and norit ...... 63 B. Clarification during acid hydrolysis. 61\. C. Filtration of dextran...... 6I4. Depolymerization...... 65 Autolysis ...... •• 66 Acid hydrolysis ...... 67 Effect of acid concentration and time ... 67 Effect of temperature ...... 70 Influence of degree of hydrolysis on distribution of fractions and their yields ...... 7 k Effect of dextran percentage in aqueous solution on distribution of the degraded polymer ...... 7J4. Molecular weight distribution . •...... 7& Ultrasonic depolymerization ...... do Procedure ...... 50 Effect of dextran concentration ...... 53 Effect of the molecular weight of dextran 57 Effect of calculated energy output .... 90 Molecular weight distribution 91 Comparison of Degradation Processes ...... 95 Integral distribution curves ...... 98 Differential distribution curves ...... 101

DISCUSSION...... 106 Factors Affecting Dextran Formation ...... 106 Clarification of Dextran ...... 107

-iii- 31 10-12 C and then refrigerated overnight.

(1+) To obtain the precipitated fraction, the solution was cooled quickly to 0t 2 C to cause the precipitated frac­ tion to adhere to the bottom of the bottle where the frac­ tionation took place. The unprecipitated fraction was de­ canted easily, as the precipitated fraction, which contained less alcohol, was a semi-solid due to partial freezing. A cloudiness or a pseudofraction appeared on cooling at i+ C 1 but did not precipitate. On subsequent warming of the sep­ arated supernatant solution to 10-12 0, the cloudiness dis­ appeared.

(5) By measuring the volume of the supernatant solu­ tion, the amount of alcohol necessary to precipitate the de­ sired fraction was calculated.

(6) Lower molecular weight fractions were obtained by repeating steps 2-5 on the supernatant solutions from the preceding fraction of higher molecular weight.

(7) Each fraction was redissolved in hot water (50 C) and the dextran was precipitated, dried and weighed as pre­ viously described. Each fraction was subfractionated in the same manner as outlined in steps 1-6.

Viscosity measurements. The viscosity of a solution is a guide to the molecular sizes of the colloids which it con­ tains. In general, the higher the viscosity, the larger the molecules. Bull et al (191+9) reported that with mixtures of 32 different sizes, viscosity is liable to be unduly affected by large molecules. It thus yields reliable results only ■when the distribution of molecular sizes around the mean is similar in the solutions being compared. All viscosity mea­ surements were made at 25 C £ 0.05 C in a water bath using Fenske modified Ostwald viscosimeter. The relative viscosity is represented by the following equation:

«{ rel. - l / y j 0 where ^ is the viscosity of the solution, and'^0 is the vis­ cosity of water. To compare the mean molecular sizes of different batches of dextran, the relative viscosity measurements were made on the 6 per cent aqueous solution using a No. 200 viscosimeter. The intrinsic viscosity measurements were made on all the fractions and the subfractions of dextran using a No. 50 viscosimeter and concentrations between 0.2 and 0.6 g/dL

(per cent). The relative viscosity, 'V/'>J0 was measured at various concentrations for every sample, using distilled water as a solvent. From the relative viscosity, the spe­ cific viscosity:

*1 sp * ^ / % - 1 was obtained. The quantity ^ sp/c ("c" in weight per cent) was calculated and plotted vs. "c", from which the Intrinsic viscosity was determined, as recorded in table 5 and figure 1 for dextran fractions. TABLE 5

Yiscosity values (*lsp/c) for fractions'"' at different concentrations (c), in distilled water

%**- Fraction ^ sp/ c ^ sp/ c n sp/c n sp/c 'n sp/c »?sp/c IPA No. c»0.6 c-0.5 c"0.i^ c=0.3 c=0.2 c=0

O.lj.2 0.1+2 0.i|l O.i+O 32.5 Fi 0.1^3 0.39

35.0 f 2 0.31 0.31 0.30 0.30 0.29 0.29 0.26 0.26 0.26 0.26 37.5 f 3 0.27 0.27 40.0 0.25 0.25 0.24 0.25 0.21+ 0.22+ FU 0.21 0.21 0.20 0.21 0.20 0.20 ij.2.5 f5 0.18 i+7.5 f6 0.18 0.17 0.17 0.17 0.17 53.8 F? ■ 0.10 0.11 0.10 0.10 0.09 0.10

Acid hydrolyzed dextran 683.

Isopropyl alcohol was used for fractionation. uoyvjfzrdD z/o3 % 09V OSV Otv 0£V ozo o/v “I i I------1------1------r

V o------o------a ------o------o — ------

(^— O' ^ 1 1 ^y ■ ■' ■" o « — ■ —

V O- V O- XL V or

y O ------£89 ?7P^X2p p-dlAfOjpfaf p pn p j vd ( lv j -- y j mv m i p u p j w i JOJ toA jm Xf /royf/A oijpzds' jvjniiy 35

The intrinsic viscosity is defined as:

I * 0 a (* lsp /c) cS.o

Light-scattering. The theory of scattering of light by solutions was presented by Debye (191+1, 191+6), in a form which permits determination of the molecular weight of a solute from measurable optical quantities, the turbidity and the specific refractive increment of its solutions.

The fundamental principle of this method may be stated somewhat as follows. When a beam of light impinges upon matter, the electric field associated with the light induces in the matter periodic oscillations of the electrons of the material. This induced oscillating electric moment w ill then emit light of the same frequency as the incident radiation so that the particle will be a secondary source of light. Its intensity is dependent on local fluctuation in concentration of the matter in the solution and on the change in the index of refraction connected with a change in concentration. Prom this intensity, the turbidity, -f , can be evaluated from scattering measurements at 90 degrees to the primary beam.

Debye showed that when the reciprocal of the specific turbidity, namely, c /f, of a dilute polymer solution in a pure solvent is multiplied by a constant H, characteristic of scattering system, and the productH {c/i) i s p l o t t e d against the polymer concentration, in the solution, a linear 36 relationship is obtained. The intercept, or the value of

H(o/'X ) for infinite dilution, should be the reciprocal of the weight-average molecular weight. H( c/'f ) a 1/M / 2Bc

- 1 / h ’where H is the refraction constant, I-i is the solute "weight- average molecular weight, 3 is a. constant depending on the

solvent and "c" is the concentration in grams per milliliter.

The turbidity, 1 , is determined by a “working standard" method, Brice et al (l?pO), a ratio of two nearly full-scale deflections is determined: One, Ors, is observed for the scattering solution at 90 degrees; the other, Gw, is observed at 0 degree, (plus neu­ tral filters) and the scattering cell is vievied by trans­ mission. The turbidity is proportions.! to Gs/G-w, end the final equation as reported by Brice et al (1950) is:

f* = I6nf Turna , Rw . , G s ■ 3 ( 1 .0 ip5 ) h ' h e * ^ Gvj ' where i is the turbidity due to concentration fluctuation, 1. is the refractive index of the solvent, T is the dif­ fused transmittance of the opal glass reference standard, I) is a diffusor correction factor, "a" is the working stand­ ard constant, F is tho product oftransmittmces of the neu­ tral filters, "h" is the depth of the beam, the factor l.Oi+5' is a correction for reflection of the primary beam, at the emergent face of the cell and Rw/Rc is a correction for in­ complete compensation of refraction effect. 37

Numerical value for constants of the turbidity equation are assembled in table 6.

The instrument used was the B-S light-scattering pho­ tometer designed by Brice et al (1930), The refractive in­ dex increments were determined with the B-S Differential

Refractometer. Slight amounts of dust particles scatter light considerably and give rise to erroneous results. Con­ sequently the dextran solution was filtered through an ultra- fine sintered glass filter into a dissymmetry cell and light scattering measurements were made at 0,4 .3 I, 90 and 133 de­ grees for both blue and green light to obtain values for both turbidity and dissymmetry. No corrections for depolari­ zation of the scattered light were made in this study.

Twenty different samples of fairly high homogeneous type dextran were used in these light-scattering measure­ ments. They were fractions and subfractions of samples pre­ viously depolymerized by different procedures, namely: acid, autolysis enzyme and ultrasonic. Turbidity and dissymmetry for a series of concentrations for a representative sample

(F^,d acid hydrolyzed dextran683)3 are shown in table 7 and figure 2. The intrinsic viscosities of these twenty samples and their weight-average molecular weights, as measured by the light-scattering, are recorded in table 8. V.'hen the molecular weights of these samples were plotted vs. the square of intrinsic viscosity, figure the data conformed to a straight line, over a range of 20,000 to 180,000. The -38-

table 6 /

Calibration data for turbidity equation

Quantity Symbol W ave Length l4-36m u. 5^6m h .

Apparent diffuse transmittance T 0.288 0.325 of opal glass standard

Diffusor correction D 0. 81(_0 0.862 Width of diaphram near cell, cm. h 1.20 1. 20 Working standard constant a 0.103 0.128 Residual refraction correction Rw/Rc 1.023 1.025 for water Neutral filter transmittances 0 .14.68 0.535 F for Filter No. 1 p i

No. 2 F 2 0.225 0 .251*. 0.100 No. 3 P 3 0.133 N o . 1*. 0.0283 O.OI4.I3 TABLE 7 Light-scattering data (turbidity and dissymetry) for a series of concentrations of dextran, using the subfraction Fc;,d

W a "/ v Cone. 4 in 0° 90° 45° 13 5 ° ^ x 103 Y x 10- (c/-f)102 Zn % iM uncorr. corr.

_»»_ y a « 0.0000 546 39.6 0.9 4.5 1.6 0.066 436 42.9 ... 1.4 6.0 3.2 0.084 0.0803 546 40.5 2.0 7.5 4.0 0.144 0.078 10.29 1.2 436 41.5 4.7 13.6 10.0 0.293 0.209 3.84 1.1 0.1576 546 40.2 3.0 9.8 6.0 0.219 0.153 10.30 1.2 436 42.8 7.6 20.0 16.0 0.459 0.375 4.18 1.1 0.2320 546 40.5^ 4.0 11.5 7.8 0.290 0.224 10.35 1.1 436 41.5 . 10.4 . 26.2 21.2 0.649 0.565 4.16 1.1 0.303? 546 41.5 5.0 14.4 9.6 0.3539 0.287 I0.58 1.2 436 43.2 "■13.0 5 -.T 32.5 26.4 0.779 0.695 4-36 1.1 0.3727 546 41.5 16.2 11.2 0.4107 0.344 IO.83 1.2

436 43.2 15.5 38.5 32.2 0.929 0.845 . 4.41 .. 1.1 0.4392 546 41.0 6.6 18.8 13.0 O.li-73 0.407 10.79 1.2 436 43.7 17.8 M-.9 35.8 1.05k 0.970 4.52 1.1 ~f x 103 Gs 90° , 7 "f » turbidity of the solution uncorr. Gw “0°~x 10^ x k corr. minus that of the solvent. Kb = 0.00295 for filter 4,1 r"‘ Zn= criterium for dust. Kq = 0.0029^ for filter k.,2 ''""“'Solvent » distilled water Figure 2. Plot f<^)xioz vs. concentration for the sub-fraction Fs, d

/o

8

z

QUO 0.20 0 . 3 0 OSO % Concentration TABLE OP CONTENTS (Continued) Page Depolymerization ...... 108 Physico-chemical Properties ...... 110 SUMMARY ...... 113

BIBLIOGRAPHY ...... 117

AUTOBIOGRAPHY ...... 1 2 k

-iv- 1+1 slope of this curve was calculated and the equation repre­ senting the relationships between intrinsic viscosity and molecular weight was found to be:

M - l.Ij-2 x 1 0 6 x [ V ]2

All the M values of these different fractions followed this relationship except those of ultrasonic depolymerized sub­ fractions, which fall just a little above the curve. This may be due to a fundamental difference in the flexibility and configuration of the dextran molecule. The starting material before depolymerization is in all likelihood made up of large rod-like molecular "coils" of relatively large cross-sections. Within the^e molecules are the dextran chains which are folded in a compact arrangement. It is believed that acid and autolysis cause these chains first to unfold and be broken at random intervals along the chain. It is proposed that ultrasonic treatment of these rod-like molecules, fractures the chains at positions other than the ether linkages between glucose units. This explanation is consistent with the experimental observation that there was no increase of the reducing power of the ultrasonically de­ polymerized dextran as compared to those hydrolyzed by au- tolysis enzyme and acid. TABLE 8

Intrinsic viscosity values and the corresponding ■weight-average molecular weights^'of some dextran fractions and subfractions

Type of Strain Sample Wt.- ave hydrolysis No. No. w m 2 mol. wt.

Acid 683 F 2,b 0.36 0.1296 179,000 tt F 2, c 0.0961 135,000 it 0.31 F 3 ,c 0 . 28 0.0676 100,000 m 0.22 0. 01+81+ 69,000 ti Fg,d 0.19 0.0391 1+5,000 11 F3,g 0.17 0.0289 1+3,000 11 F6*f 0. 1L|. 0.0196 27,500 Acid 512 Gl,b 0.30 0.0900 127,000 it G-li c 0. 22 0. 01+81+ 70,000 it G 3,a 0.17 . 0.0280 1+2,000 11 31,500 it G3 , b 0.15 0.0225 G l+>a 0 . iii- 0.0196 27,000 ti G3 ,c 0.12 0 .011+!+ 21,000

Autolysis 512 Ei,b 0.58 0. 336Li- 500,000 11 E]_,d 0.35 0.1225 175,000 it e 3 0.27 0.0729 100,000 11 EJ* 0.18 0.0321+ 1+8,000 Ultrasonic 512 V lfc 0.275 0.0756 116,000 ii U 2 ,a 0.27 0.0729 lli+,000 n u3 0.25 0.0625 105,000

As measured by the light-scattering

Weight-average molecular weight j7?o/ecu/ar weight x/o~‘ to /z e / o.oz gur . o m/c/r iht vs. t eigh w mo/ccu/ar f o /ot P 3. re u ig F 0.04 rai, v/scost triasic, m ati i vsast sguare>. sity v/sca sic tria ia JLl-3 JO O O/z EXPERIMENTAL

Factors Influencing Dextran Fermentation It has been reported by many research workers that many factors seem to influence the yield, the properties and the by-products during the active fermentation of dextran. The

following experiments were conducted to investigate the ef­ fect of various factors on the production of dextran.

Effect of strain and nutrients. Six large Erlenmeyer flasks, each containing five liters of media (three having media No. 1, the other three, media No. 2), were inoculated with active cultures of Leuconostoc me senteroides 683* B-512 and L. dextranicum "elai"; i.e., each strain was inoculated into the two comparative media. The flasks were incubated

at 25 C for five days after which the fermented media were clarified and the dextrans in each flask were precipitated, dried, weighed and the per cent yields were calculated. The

results were recorded in table 9, indicating that the strain

as well as the constituent nutrients in the media has a marked effect on the yield and that medium No. 2 is more satisfac­

tory for the production of dextran on a large scale than is medium No. 1.

The properties of the types of dextran produced were examined. It was noticed that during the fermentation of

L. mesenteroides 683, the dextran formed, settled to the

1 k S

TABLE 9

Effect of media* and strain of Leuconostoc on the yield of dextran

Percentage yield*'" using Strain Media No. 1 Media No. 2

683 68.0 75.0

B-512 53.0 68.0

"elai,T i{-2.0 66. 6

The Ingredients of these media were previously reported. Glucose available from sucrose. 4 6 bottom of the flask in a very viscous layer, which, in turn, contained some dextran in the form of hard masses or clumps. When the flasks -were manually shaken, all of the viscous layer went into solution, but the clumps did not. Upon standing, the viscous layer settled again. This character­ istic might be of value, especially in large scale produc­ tion. L. mesenteroides B-$12 produced a polymer which was much better to handle; it can be clarified and precipitated more easily than that of 683 and "elai". Strain "elai" pro­ duced a type of dextran which was very difficult to clarify and to dissolve in water. This noticeable difference in properties might be due to the inherent structure of the polymer.

Effect of extended incubation. An experiment in the form of serial analyses to follow the dextran fermentation on prolonged incubation using different strains on media No. 1 and No. 2 was performed. In the previous experiment it was noticed that the fermented liquor of Lj_ mesenteroides 683 was not homogeneous; it contained some clumps of dextran. Hotvever, in order to avoid error in taking representative samples for. serial analysis of yield, small Erlenmeyer flasks containing media No. 1 and No. 2 were used, so that, at different periods of time serial analyses were carried out using the entire contents of the flasks. Fifteen flasks of each media were inoculated with strain 683 and strain 1+7 B-512, which, were incubated at 25 C. The serial analyses were made on the fermented media in order to follow the ef­ fect of extended incubation on: a. The total acid production. b. The pH. c. The dextran yield. d. The viscosity of 6 per cent aqueous solution."' Duplicate experiments were conducted and the average result are tabulated in tables 10, 11, 12 and 13. when these re­ sults were plotted vs. time, as shown in figures i+, and 6, the following observations were made. (1) A sharp drop in the pH of all the fermented media occurred during the first 36 hours of incubation, followed by a slight further decrease up to 110 hoiirs, after which a steady value of 3*8 was reached. (2) The amount of total acids.increased, following ex tended incubation up to 72-110 hours after which no measur­ able increase was detected. (3) The relative viscosity measurements of 6 per cent aqueous solution of dextran reached a maximum at about 36 hours followed by a sharp decrease which slowed down after 110 hours. An interpretation of the decrease of viscosity on prolonged incubation was established when the results of the total acids and the relative viscosity are plotted

“The dextran was not clarified. 4 8

TABLE 10

Serial Analyses Data Effect of extended incubation on the dextran fermentation, using Leuconostoc mesenteroldes NRRL B-512 on unbuffered media Medium No. 1

T ime in PH T. acids '* Rel. vis. hours value m/MlOO cc. yield of b>% soln.

12 6.50 3.62 33.3 __

24 5.30 6.9 5 36.6 44.1 36 4.18 9.39 44.4 46.5 4.00 10. 28 53.2 37.2

58 3.85 11.23 45.0 27.8 72 3.80 11.54 35.0 26.0 110 3.75 13.30 30.0 22.0 135 3.75 13.30 30.0 17.4 185 3.75 13.30 30.0 9.1 234 3.75 13.70 27.0 8.3 282 3.75 13.70 25.4 8.2 360 3.75 13.70 23.2 8.1

Total acids in millimoles per 100 cc. of fermented media. Percentage yield based on the glucose available from sucrose. Relative viscosity of 6 per cent aqueous solution of dextran. Not tested. lj-9

TABLE 11

Serial Analyses Data

Effect of extended incubation on the dextran fermentation> using Leuconostoc me senteroides 683 on unbuffered media

Medium No. 1

T ime in pH T. acids 70Of Rel. vis. of hours value mM/100 cc. yield 6% soln.

12 6.35 3.50 13.2 26.7 2l+ If-.90 8.04 I4.6 .6 169.3 36 1+.10 9.83 14,8.8 129.6 it-9 3.90 10.95 67.8 127.0 58 3.90 11.30 58.1+ 125.0 72 3-90 11.30 53.2 62.0

110 3.85 11.30 52.2 1+1+. 1 135 3.85 11.30 51.0 36.0 185 3.85 11.30 50.0 30.2 23^ 3.85 11.30 50.0 26.1 282 3.80 11.30 50.0 20.9 360 3.80 11.30 50.0 20.0 £0

TABLE 12

Serial Analyses Data

Effect of extended incubation on the dextran fermentation, using Leuconostoc mesenteroides NRRL B-512 on unbuffered media Medium No. 2

T ime in pH T. acids % Rel. vis. of hours value mM/lOO cc. yield 6u/o soln.

12 6.10 3.63 28.2 20.0

24 14-. 65 7-1+3 35.0 25.0

36 4.10 8.83 ll-6.0 50.0

k9 1+.10 9.11-5 65.0 56.0

58 3.95 9.65 68.0 36,0

72 3.95 10.06 65 • 0 24.O

110 3.80 10.62 63.0 22.0

135 3.80 10.62 62.0 20.0 185 3.80 10.79 60.0 18.0

234 3.80 10.79 58.0 16.0

2 3 2 3.75 . 10.811- 56.0 14.0 360 3.75 11.07 51.0 13.0 LIST OF TABLES TABLE PAGE

1. Average per cent recovery of a lj.#399l4. per cent solution of dextran 6 8 3 using various concentrations of precipitating reagents •••••• 19 2. The effect of humidity on dextran's weight .... 21 3« Determination of the "k" constant for the mea­ surement of concentration of dextran, using the Dipping Refractometer...... 21}. 1}.. Measurement of percentage concentration, using the Dipping Refractometer «...... 25> 5>. Viscosity values ( i|sp/c) for fractions at dif­ ferent concentrations (cj In distilled water .. 33 6* Calibration data for turbidity equation •■•...• 38 7. Light-scattering data (turbidity and dissymetry} for a series of concentrations of dextran, us­ ing the subfraction F ^ , d ...... 39 8. Intrinsic viscosity values and the corresponding weight-average molecular weights of some dex­ tran fractions and subfractions ...... ij.2 9* Effect of media and strain of Leuconostoc on the yield of dextran...... l\.$ 1(J* Serial analyses data - Effect of extended in­ cubation on the dextran fermentation, using Leuconostoc mesenteroides NRRL B-^12 on un- buffered medium No. I ...... lj.8 11* Serial analyses data - Effect of extended in­ cubation on the dextran fermentation, using Leuconostoc mesenteroides 6 8 3 on unbuffered medium No. 1 ...... J4.9 12. Serial analyses data - Effect of extended in­ cubation on the dextran fermentation, using Leuconostoc mesenteroides NRRL B-i?12 on un- buffered medium No, 2 • • ...... 5>U

-v- 51

TABLE 13

Serial Analyses Data

Effect of extended incubation on the dextran fermentation, using Leuconostoc mesenteroides 683 on unbuffered media

Medium No. 2

T ime in pH T. acids % Rel. vis. of hours value rnM/lOO cc. yield 6/6 soln.

12 5.30 6.15 23-8 20.6

2 k I4..8O 9.61 32. 2 30.0

36 1+.50 10.17 68.0 50.0 ^9 1^.15 10.77 65.0 36.2 58 J+.15 10.90 6J4..O 35.0 72 I+.05 11.30 60.0 31.6 110 3.85 11.30 59.6 28.6

135 3.85 11.30 58.0 20.6 185 3.85 11.30 56.0 19.7

23^ 3.80 11.30 55.0 19.0 282 3.80 11.30 52.0 18.5 360 3.80 11.30 50.0 16. 6 R elation_ between tim e ond^ pH durintj thel deztcarr fermentation usin^ different strains, or compara tive*„. me dig.

G&3 on medium, nai

O --- sol on medium aoj ft 6a3 on medium ng? 3/2 on medium no?.

!8====0-=--Q= ------0

_J______I______I------1------L J?n /no 24-0 200 3£o Time, in hours 1 4 0

l ^ 120 r I& 0—o- 0-. -0 0 I • ------•—• ------1 m 'it > * 683 on medium, aoj — 0— Six on. medium no.! ^ VjJ

1 fo — # — 6 6 3 o i l m e d i u m n o z

6 0 -II 2 — • — 512 on medium no.z \ $ 40 V\ Figures, gelation between time and total acids in dextran. fermentation, using different Strains on comparative, media.

I______I______I______I______I__ 6 0 /ZO /SO 240 300 3 6 0 Time, in hours. 2 0 0 - FiqurcG. gelafion bet.weea.dime undrelative^ -8 viscosity of 6% aqueous so/u tion of dextran. produced, by-different. strains on-companative media. H 633 jojl medium no. I

X ^ 2 0 — O - - sn —oa medium na/

+ -- 633 011 M 2-ediaaLJiQ2 t § so — • - - r/? on medium m2 ^ i 0 40

ZO -' I

60 120 130 240 3 0 0 Time in hours 55 together against time (figure 7)» the marked decrease of viscosity coincided with the sharp rise of total acid. Gard­ ner (1952) stated that the acidity of the culture media has no effect on the viscosity of dextran. Thus, it can be stated that the acidity of the culture media will enhance the liberations of an autolyzed enzyme after death of the Leuconostoc cells, which will degrade the polymer on ex­ tended incubation. (i^J There was a sharp increase of percentage yield of

dextran which reached a maximum value at l±9 hours. This was followed by a marked decrease up to 72 hours, after which no measurable changes were detected. This might be explained on the basis of the degradation of dextran through autolysis, which forms a lower molecular weight fraction that is not precipitated when 6u per cent isopropyl alcohol is added for the precipitation of the polymer.

Effect of mechanical shaking. The effect of mechanical shaking at room temperature on the dextran production using Leuconostoc mesenteroides 6d3 and B-512 on the two compara­ tive media was investigated. The procedures for the prepara­ tion of the flasks, their inoculation, and the serial analy­ sis investigation were the same as the previous experiment except that the flasks were placed in a mechanical agitator during the incubation period. The results as shown in tables lif. and 15 indicate the following: 56 7/OJjf?/o£ jo Xj/roDr/A

§ § ■ * o T T VO T T T

o I -I Si I

§ 1

Q) Q?

I U S in in h o u rs

\

o VO

• # X 1 i ^ «D vo ^ ZVJ>?1£/ p3jVJU/J3J/Z£/007^p7D tr_Z pf W 57 (1J A drop of pH value occurred during the first £3 hours after which no measurable change took place*

( 2 ) There was an increase of total acids up to £3 hours after which no measurable increase was detected. (3) There was a slight increase in the relative vis­ cosity of dextran produced by 6b3 followed by a slight de­ crease* (Z+J The yield of dextran using both strains on the com­ parative media was very poor, indicating that the mechanical shaking is unfavorable for dextran production*

Effect of neutralization. The effect of neutralization of acids produced during the early hours of the dextran fer­ mentation, on the yield and the type of dextran was investi­ gated. Two large flasks, each containing 10 liters of media No* 2, were inoculated with L* mesenteroides B-512, using 5 per cent active inoculum which had beenneutralized prior to inoculation. The flasks were incubated for 5b hours at 25 C, and were examined every 3 hours by withdrawing a sam­ ple for pH measurements. An alkali (5N NaOHJ was added to one flask only, to maintain the pH at 6.£. This neutrali­ zation was made for the first 21+ hours of incubation. The fermented media were clarified and the dextrans were recovered, dried and weighed. This experiment was duplicated and the results are:

(1) There was an active fermentation and a noticeable TABLE lij.

Serial Analyses Data

Effect of extended incubation and mechanical shaking on the dextran fermentation, using L. mesenteroides NRRL B-512 and 683 on unbuffered media

Medium No. 1

Time L. mesenteroides B-512 L. mesenteroides 683 in pH T.acids % pH T. acids % Rel.vis.of hours value mM/lOO cc yield value mM/lOOcc. yield 6% soln.

27 6.0 6.9 Trace 5.5 9.5 3 4 4 12.0

h 53 4.7 10.6 k -7 9.6 18.0 12.8 ti 75 k - k 11.0 k -7 9.6 20.0 13.0 11 135 k - k 11.5 k -k 9.7 31.0 llj-.O ti 23k k - k 11.5 k -3 10.6 20.0 15.0 11 360 k - k 11.5 k -3 10.6 19.6 llp.7 TABLE 15

Serial Analyses Data

Effect of extended incubation and mechanical shaking on the dextran fermentation, using L^ mesenteroides NRRL B-512 and 683 on unbuffered media

Medium No. 2

Time L. mesenteroides B-512 L. mesenteroides 683' in pH T.acids 1o PH T.acids % Rel.vis.of hours value mM/100 cc yield value mM/100 cc yield 6% soln.

27 5.2 5.8 Trace 4.8 8.7 6.6 12.0 it 53 k-9 6.7 k-3 9.7 22.2 17.0 ii 75 k-3 7.1+ k-2 9.7 20.0 16.2 it 135 k-3 7 4 k*o 9.8 15.0 15.6 ti 231. k-3 7 4 k-o 9.8 11.0 15.0 it 360 k-3 7 4 k-o 9.8 10.8 4 . 0 60 increase in viscosity of the fermented liquor in the neu­ tralized media. (2) A larger yield of dextran was obtained from the neutralized culture, as shown on table 16. (3) A 6 per cent aqueous solution of the neutralized dextran had a relative viscosity of lj.5.0, and that of the untreated dextran was 23*0. This difference in viscosity might be due to autolytic degradation of the untreated dex­ tran which is favored by the acid production as explained in the previous experiment. {Lp) The higher yield of dextran (85 per cent) in the neutralized culture, as compared to 67 per cent in the un­ treated culture, may be explained on the basis of the opti­ mum pH for the production and the activity of the dextran synthesizing enzyme. Koepsell and Tsuchiya (1952) reported that pH is a critical factor affecting the production of dextransucrase, the optimum being about 6.5-7*0, in the growing culture. It seems that periodic neutralization to keep the pH at 6.5 during the early hours of fermentation will favor the enzyme production. This increase in enzyme production results in a higher yield of dextran, since it is produced under the conditions most favorable for dextran synthesis (pH 5-0-5*2) as stated by Koepsell and Tsuchiya

(1952) . LIST OP TABLES (Continued) TABLE PAGE 13* Serial analyses data - Effect of extended in­ cubation on the dexti'an fermentation, using Leuconostoc mesenteroldes 683 on unbuffered medium No* 2 ...... 51 lij.. Serial analyses data - Effect of extended in­ cubation and mechanical shaking on the dex­ tran fermentation, using L. mesenteroides NRRL B-512 and 683 on unbuffered medium No.l .. $ 8 15* Serial analyses data - Effect of extended In­ cubation and mechanical shaking on the dex- tran fermentation, using L. mesenteroides NRRL B-512 and 683 on unbuffered medium No.2 •• 59 16. The effect of neutralization on the dextran production...... 61 17* Fractional precipitation of two liters of 2 per cent aqueous solution of autolyzed dextran B - 5 1 2 ...... 68 18. Molecular weight distribution of autolyzed dextran B-512 ...... 69 19* The effect of acid concentration and time on the hydrolysis of dextran ...... 71 20. The effect of temperature on the rate of acid hydrolysis ...... *...... 73 21. The influence of the degree of. hydrolysis on the yield of fractions precipitable by various concentrations of Isopropyl alcohol ...... 75 22. The effect of dextran concentration In aqueous solution on the distribution of the different fractions...... 77 23* Fractional precipitation of 100 ml of aqueous solution of acid hydrolyzed dextran...... 78 2 If., Fractional precipitation of two liters of aqueous solution of acid hydrolyzed dextran 683 ...... *...... 79 -vi- 61

TABLE 16

Tlie effect of neutralization on the dextran production

Time in Neutralized culture Regular culture hours % „ rel. 7o rel. pH'::" yield'** vis pH yield vis.

0 7.20 No dextran 7.20 No dextran 3 6.70 re covered 6.70 recovered

6 5.14-0 --- -- 6.20 ------

9 5.70 --- -- • 5.60 — _ - — 12 5-14-0 ------5 .0 0 ------15 5 .2 0 -- -- 1+.70 --- --

18 5 .2 0 -- -- U-.li-O -- -- 21 5.20 -- -- i+.OO -- -- 2k 5.oo --- -- 3.90 -- -- 5o I4..2O 85.0 14-5.0 3.80 67.0 23.0

pH value before neutralization to pH 6.5 using 5 N NaOH, during the first 2l\. hours of incubation. Percentage yield of dextran calculated on the basis of glucose available from sucrose. Relative viscosity of 6 per cent aqueous solution of the recovered dextran after 50 hours incubation. 62 Clarification of Dextran Dextran may be water-soluble, partially soluble or in­ soluble, depending upon tbe strain of the organism which pro­ duces it, Leuconostoc mesenteroides B-512 and 683 produce a water-soluble polymer, strain 523 produces a water-insolu­ ble material while L . dext rani cum "elai" produce a partially soluble dextran. An aqueous solution of a water-soluble dextran has a faint opalescence which varies greatly from a white cream color to a blue tinge. This opalescence might give an un­ desirable appearance for a plasma volume expander. The fol­ lowing experiments were conducted to investigate the cause for this opalescence, and to establish a quick, reliable procedure for the dextran clarification.

Dextran opalescence. Stained smears and hanging drop slides were made from an aqueous solution of unhydrolyzed dextran produced by L. mesenteroides 683- Bacterial cells, which were found to be present, were counted and an averagp value per field was determined. The aqueous solution was centrifuged for several hours, after which a noticeable de­ crease in the opalescence was detected. The supernatant was decanted and the white residue recovered, from which stained smears and hanging drop slides were made. Bacterial counts were made from both preparations and an average value per field was determined and the following information was obtained: (1) The supernatant had a bacterial content consider­ ably lower than the original aqueous solution of dextran. (2) The residue was found to be mostly bacterial cells, yet smaller in size (0.5-0.2 u in diameter) than the normal cells of Leuconostoc cultures (1.0-0.8 u). (3) When these bacterial cells were resuspended in water, a white opacity was noticed comparable to that of the original aqueous solution of dextran. It can be stated that the opalescence was mostly due to the bacterial cells. An interpretation based on these results is that bac­ terial cells of Leuconostoc) being living organisms, have protoplasm containing protein, which may be physically at­ tached to the polysaccharide dextran molecules, leading to the characteristic opalescence of the aqueous solution.

Methods of clarification of dextran. Prom the previous experiments it w a s •established that the protein of the bac­ terial cells, in physical attachment to the dextran molecule causes the opalescence of aqueous solutions of dextran. To achieve clarification, the bacterial cells should be removed from the dextran. In this experiment several methods were tried to eliminate the cells.

A. Use of super-cell and norit: Activated charcoal (norit) was added to a 5 pei* cent aqueous solution of crude dextran, heated to 80 G and filtered hot through a pad of 64 super-cel in a Buchner funnel under vacuum. Various thick­ nesses of the super-cell pad were used to establish the most i efficient thickness. Clarification was accomplished but it was noticed that the super-cell adsorbed a considerable amount of dextran from the solution.

B. Clarification during acid hydrolysis: One hundred- twenty grams of crude dextran were partially hydrolyzed in two liters of 0.1N HC1 at 60 C in a water bath. The hydroly­ sis was followed by viscosity measurements and was stopped by neutralization when the relative viscosity reached a value of 2.8. The solution was filtered through a Berke- feld filter and the dextran recovered. It was noticed that the opacity of the dextran solu­ tion was markedly reduced during hydrolysis and completely absent after filtration. It seems that -the 0.1N acid de­ stroys the adhesive forces between the dextran molecules and the protoplasm of the bacterial cells and thus dissolv­ ing the opacity-forming material, while filtration through the bacterial filter totally eliminates the opalescence.

C. Filtration of dextran: Seitz and Berkefeld fil­ ters were used to eliminate the bacterial cells from the aqueous solution of 6 per cent crude dextran. It was found that a repeated filtration was necessary to clear the dex­ tran solution as the bacterial cells could pass through the filter. It seems that the dehydration process of the crude 65 dextran by repeated precipitation with, alcohol, probably caused the bacterial cells to shrink to such a size that they can pass through the filter. Finding that the dehydration procedure caused the bac­ terial cells to shrink and pass through the bacterial fil­ ters, it was deemed advisable to clarify the fermented cul­ ture media before precipitation of dextran. The fermented culture media was diluted with distilled water, using 20 per cent by volume, and filtered through large bacterial filters (capacity - two liters). The dex­ tran was precipitated and it was found that an aqueous so­ lution of this dextran was clear. This method proved to be quick and efficient.

Depolymerization of Dextran The future of dextran as a blood volume expander is de­ pendent upon the molecular weight and its homogeneity. In- gslman and Hailing (19^-9) established that the high molecu­ lar weight dextran enhances the sedimentation tendency of the red blood-corpuscles and that the lower the molecular weight of the polymer, the less is this action. In the production of such a material for the use as a blood extender, it is necessary either to control the fer­ mentation to produce the desired molecular size or degrade the high molecular weight dextran to a value where the ma­ jority of the degraded polymer will have molecular weights 66

in the order of the plasma protein (about 75,000 6 25,000) as reported by Pulaski (1952). This molecular weight should be low enough to give a high colloidal osmotic pressure and high enough to enable the dextran molecules to stay for some time in the blood vessels without being immediately filtered through the glomerular membrane of the kidneys and through the walls of the blood capillaries as stated by Ingelman and Hailing (19i|-9). The following experiments were conducted to produce these desired molecules through degradation using different procedures. Factors affecting the degradation was investi­ gated followed by subsequent studies on the hydrolyzed ma­ terial using fractional precipitation with the aim of pro­ ducing a dextran polymer having a physico-chemical proper- - ties of a plasma volume expander.

Autolysis

Production of a low molecular weight dextran was accom­ plished through extended incubation beyond the time of maxi­ mum viscosity. The following studies were carried out to investigate whether autolysis degradation results in some selective structural change in dextran, or merely produces a random decrease in molecular size. Two liters of 2 per cent aqueous solution of low molecu­ lar weight dextran B-512 (['*(]s 0.33 / 0 .05) were subjected to molecular weight homogeneity test. Five main fractions were -67- obtained (E-^-E^) at the following alcohol concentrations: 32.5, 37*5* 1+2.5> 1+7.5 and 55*0 per cents. The percentage yields of the fractions were calculated and their intrinsic viscosity were made. This experiment was duplicated and the average results are given in table 17. The first two frac­ tions (E^ and E2 ) were further refractionated into subfrac­ tions, a, b, c, etc. Intrinsic viscosity measurements were performed on all the subfractions while light-scatter­ ing experiments were carried out on some of the fractions and the sub-fractions. The results are presented in table 18, which indicate that autolysis results in a random deg­ radation in molecular size.

Acid Hydrolysis

It has been reported by Ingelman and Hailing (191+9) and Renfrew and Cretcher (191+9) that production of molecules of desired molecular tiieight can be accomplished through partial acid hydrolysis. Studies using partial acid hydrolysis were made to investigate the effect of certain factors on the deg­ radation and the molecular distribution.

Effect of acid concentration and time. A 6 per cent solution of high molecular weight dextran B-512 was hydro­ lyzed in different concentrations of hydrochloric acid (0.05, 0.10, 0.15 and 0.20N) at 80 C. in a water bath. The hydrolysis was followed by relative viscosity measurements 68

TABLE 17

Fractional precipitation of two liters of 2 per„cent aqueous solution of autolyzed dextran B-5>12'"

% Wt. of % Notation VJt.- ave. “ “ IPA fractions yield of 0 0 mol. wt. in g. fractions

32.5 19.81 49.52 E1 0.48 327,000

37.5 5.81 14.52 E 2 0.36 129,600

2.73 6.82 e 3 0.27 100,000

0.18 48,000 47.5 2.35 5.87 e4

55.0 1.59 3.97 E5 0.14 27,000

This autolyzed dextran has an intrinsic viscosity of 0.33 C 0.05. Weight-average molecular weight as measured by light scattering. 69

TABLE 18

Molecular weight distribution of autolyzed dextran 5-512'“'

Fraction Sub- W t. - ave ... % 0 0 !> fraction H yield"'"'' mol. wt.

E i a 30.0 37. *1- 0.68 656,500 b 32.5 I.I4. 0.58 500,000 c 35.0 24.-O 0.50 355,000

d 37.5 3.1|- 0.35 175,000 e 14-5.0 1.3 0.25 89,000 f 55.0 1.0 0.17 14-3,500

e 2 a 35.0 10.8 0.39 21*4,000 b 37.5 1.8 0.26 96,000 j c i|-5 .o 0.8 0.20 57,000

d 55.0 0.8 0.10 114,000

Fractions E-^ and E 2 were used in this study.

-:hc- per Cent yield, based on the original weight of the fractionated dextran. Weight-average molecular weight as measured by light- scattering. 70 / of the degraded dextran at different time intervals. Table 19 and figure 8 present the results of these measurements, which indicate the following: (1) The rate of hydrolysis is directly proportional to the acid concentration and time. (2) There is a quick drop in the viscosity during the early minutes of hydrolysis using different acid concentra­ tions, indicating a high intensity of degradation.

(3) The curves of all these experiments have almost the same pattern, indicating that the mechanism for acid hydrolysis is the same for the different acid concentra­ tions used.

Effect of temperature. Hydrolysis of a 6 per cent sol­ ution of dextran in 0 .10N hydrochloric acid was made in a water bath under various temperatures, namely, lf.5 , 70, 80 and 98 C. The rate of hydrolysis was followed by viscosity measurements at 20 minute intervals. The results, recorded in table 20, shows: (1) The rate of hydrolysis is directly proportional to the temperature. (2) Considerable increase in the rate of degradation was noticed at 98 C . Prom previous experiments it can be stated that acid hydrolysis can be made to proceed smoothly under controlled conditions of acid concentration, temperature and time. LIST OP TABLES (Continued) TABLE PAGE 25. The molecular weight distribution of acid hydrolyzed dextran 683 ...... til 26* Effect of ultrasonic vibrations on dextran .... ti2 27. Preliminary test for molecular weight homoge­ neity of an ultrasonic degraded dextran ...... tilj.

28. Effect of dextran concentration on the ultrason­ ic depolymerization ...... 85

29. Effect of the molecular weight of dextran on the ultrasonic degradation...... 88 30. Effect of calculated energy output (intensity) of the ultrasonic vibrations on the degrada­ tion of dextran B-512...... 92 31* Fractionation of two liters of 2 per cent aqueous solution of ultrasonic depolymerized dextran B-512 ..... 93 32. The molecular weight distribution of ultra­ sonic degraded dextran B - 5 1 2 .... •••••...... 91+ 33. Comparison of acid and ultrasonic depolymeri­ zation of the high molecular weight dextran ... 96 3l+. Fractional precipitation of two liters of 2 per cent aqueous solution of autolyzed, acid hydrolyzed and ultrasonically depolymerized dextran B-512 ...... 99 35* The molecular weight .distribution of acid hy­ drolyzed dextran B-512 ...... 100 36'• Integral distribution data for the degraded polymers of different procedures of depolymer­ ization ...... 102

-vii- 71

TABLE 19

The effect of acid concentration and time on the hydrolysis of dextran

Time of hydrolysis Rel. vis. of 6% soln.” in various in acid concentrations minutes 0.05N- 0. IN 0 .15N 0 .2N

0 23.0 23.0 23.0 23.0

20 12.0 11.0 8.6 6.8

ko 9.0 5.7 ip. 0 3.0 60 7.5 3.7 3.2 2.2

80 5-k 3.i|- 2-k 2.0 100 I4..5 2.6 2.2 1.8 120 3.8 2.1+ 1.9 1.7 llj-0 3.2 2.1 1.8 1.6

Relative viscosity of 6 per cent solution in various acid concentrations at 80 C in a water bath. E ffect of acid concentration ao^thc hydrolysis of dexirjm

-j r\)

I 3 0 m o l z o * 4 0 ASO

T i m e 73

TABLE 20

The effect of temperature on the rate of acid hydrolysis*

Time of hydrolysis Rel. vis. of G% soln. at the in following temperatures minutes k $ 0 70 C 80 C 98 C

0 23.0 23.0 23.0 23.0

20 21 .]+ 12.2 11.0 1.7 ^0 21.2 5.8 5.7 1.3 60 21.0 i+-3 3.7 1.3 80 20.5 3.5 3.i+ 1.3 100 19.8 3.1 2.6 1.3 120 19.2 2.8 2.1+ 1.3

Hydrolysis was made in 0 .1N hydrochloric acid. Factors Affecting; tlie Molecular Weight Distribution Using Acid Hydrolysis The influence of degree of hydrolysis on the distri­ bution of the fractions and their yields. Two samples (A and B) of high molecular weight from one batch of dex­ tran B-512 were hydrolyzed in 0 .1N HC1 . Degradation was followed and stopped when the relative viscosity of sample

A reached a value of 3-02 and sample B, a value of 2 .2 0 . The degraded polymer of each sample was recovered and an aqueous solution of 5 per cent was subjected to fractional precipitation using isopropyl alcohol. No attempt was made to recover the degraded dextran which was not precipitated by 65 per cent isopropyl alcohol. The precipitated frac­ tions were dried and weighed and the percentage yield of every fraction was calculated on the basis of the initial amount of dextran. Duplicate experiments were made. Table 21 presents average results o£ this experiment and indicate that when polymers having a relative viscosity of 2.20 are fractionated, a number of different fractions are obtained.

The effect of dextran percentage in the aqueous solu­ tion on the distribution of the degraded polymer. Aqueous solution of 12, 10 and 5 per cent concentrations of this dextran from sample B were prepared. These solutions were subjected to fractional precipitation using isopropyl alco­ hol. .The yield of every fraction was calculated. Duplicat experiments were done, and the average values are presented 75

TABLE 21

The influence of the degree of hydrolysis on the yield of fractions precipitable by various concentrations of isopropyl alcohol

%■ Percentage yield for IPA Sample A'” '" Sample

30.0 -- - — 35.0 i-j.l. 8o 21.16

l+o.o 23.00 2k* 2l\. kS -o 7 .I4-O ±k*oo

5o.o 3.^0 6.60 55.0 1.80 2.68

60.0 2.80 0.80

65.0... ---- 0.52

Total recovery 80.20 70.00

Percentage of isopropyl alcohol. Sample A and B had a relative viscosity of 3.0 2 and 2 .20* respectively. Sample not recovered. No precipitation was attempted beyond 6 IPA. 76 in table 22 and indicate that better fractionation is ob­

tained when the aqueous solution contained 2.5 per cent dextran.

Molecular, weight distribution. A 2.5 per cent aqueous solution of acid hydrolyzed dextran 683'% which has an in­ trinsic viscosity value of 0 .19, was subjected to the molec­ ular weight homogeneity test as previously described. A preliminary fractionation was made on 100 ml. Iso­ propyl alcohol was added and a total of seven fractions were obtained at the following alcohol concentrations: 32.5, 35.0, 37*5, J4.O.O, 1+2.5, J+7‘5 and 53*8 per cents. This ex­ periment was repeated several times and the average results are recorded in table 23- Another experiment was performed on a larger scale.

Two liters of a 2.5 per cent aqueous solution of dextran were fractionated into seven main fractions (F^-Fy) as de­ scribed above. Intrinsic viscosity measurements were made on these fractions and the results are given in table 21}. and figure 1. All these fractions with the exception of Fy, were further refractionated into sub-fractions a; b, c, etc., at

the following concentrations: 32.5 , 35*0 , 37*5 , %0 .0 , %2 .5 , lp5.0 and 50.0 per cents. Some physico-chemical studies were carried out 011 these fractions. Intrinsic viscosity measure-

This hydrolyzed dextran was obtained from The Commonwealth Engineering Company, Dayton, Ohio. It was clarified and dried as previously described. 77

TABLE 22

The effect of dextran concentration in aqueous solution on the distribution of the different fractions

% Percentage yields using IPA 12 °/o 10% 5%

30 0.50 *vr*i r --- 35 38.50 2 6 .I4.0 21.16 k-o 21.14-0 30.20 2I4-. 2li. kS 6.00 7.14-0 II4..OO 50 l.lj.0 14-.60 6 . 60 55 3.5o 3.60 2.68 60 0.60 0.72 2.20 65 o.5 o --- 0.80 Total recovery 7 2 .14-0 72.92 70.00

Based on the original amount of dextran in the aqueous solution.

Not recovered • 7 8

TABLE 23

Fractional precipitation of 100 ml"' of an aqueous solution of acid hydrolyzed dextran

Of 0/ Alcohol /° „„ Weight of fraction P in ml IPA’* “ In grams yield

1+8.11+ . 32.5 0.1+2 16.8

5.1+6 35.0 0.1+7 18.8

5.80 37.5 0.1+7 18.8 5.85 L+0.0 0.21 8.1+ 6.52 1+2.5 0.23 9.2 11+. 1+0 1+7.5 0.33 13.2 22.50 53.8 0.10 1+.0

Containing 2.5 grams of degradated dextran 683. Isopropyl alcohol. 79

TABLE 2l+

Fractional precipitation of two liters of aqueous solution of acid hydrolyzed dextran 683"

Qt %C~*\ Alcohol Wt. of /0 Notation of in ml fraction yield fraction

962.96 8.10 g 16.20 F1 0.39

101+.23 9.1+7 g 18.81+ P 2 0.29

119.20 9.01 g 18.02 f 3 0.26 119.79 i|.06 g 8.12 Fl+ 0.20 132.80 1+.67 g 9.31+ F5 0.18

301.52 7.87 g 15.71+ f 6 0.16 L|52.00 1.92 g 3.81+ f ? 0.10

This dextran has an intrinsic viscosity value of 0.19. ':HC' Percentage yield based on the original weight of the dextran.

Intrinsic viscosity measurements at 25 G £ 0.05. 80 ments were made on all of these sub-fractions. Light-scat­ tering measurements for molecular weight determinations were performed on some of these sub-fractions. The results are recorded in table 25 and indicate that acid hydrolysis leads to a polydispersed polymer with a 26.96 per cent yield

of the desired molecular weight < M 0.23 £ 0 .0 5 ).

Ultrasonic Depolymerization

Considerable attention has been devoted in recent years to the applications of ultrasonics to industry, metallurgy, biology and medicine. Many of these applications have proved to be highly successful. This study was made to investigate the possibility of using the ultrasonic vibrations in the degradation of the high molecular weight dextran to a low - molecular weight polymer for the use as a plasma volume ex­ pander.

Procedure. A 6 per cent aqueous solution of high mo­ lecular weight dextran B-512 was subjected to ultrasonic vi­ brations for a total period of 3.5 hours using a vvoltage in­ put of 2.2 k.v. and a current input of 250 m. amps, i.e., a calculated energy output of 550 watts. Irradiation was made in 15 minute periods as it was necessary to stop the ultra­ sonic treatment to cool the crystal. The degradation was followed by viscosity measurements as shown in table 26. The degraded polymer was recovered and its intrinsic vis- LIST OP FIGURES FIGURE PAGE

- 1. Specific viscosity curves for fractions (F^-Fy) of acid hydrolyzed dextran 683 ...... $l\.

2. Plot (c/-f ) x lO^ v s . concentration for the subf raction F^, d ...... • I4.U 3. Plot of molecular weight vs. intrinsic vis­ cosity square ...... Jlj.3 [j.. Relation between time and pH during the dex­ tran fermentation, using different strains on comparative media ...... 52 5. Relation between time and total acids in dex­ tran fermentation, using different atrains on comparative m e d i a ...... 53 6. Relation between time and relative viscosity of 6 per cent aqueous solution of dextran produced by different strains on comparative media ...... $l\. 7. Relation between time, total acids and rela­ tive viscosity during the dextran fermenta­ tion, using L. mesenteroides B-512 on medium no. 2 ...... r::;;:: ...... 56 8. Effect of acid concentration on the hydrolysis of dextran...... 72 9# Effect of molecular weights of dextran on the ultrasonic degradation, using 1 per cent aqueous solution of dextran B-512 ...... 86 lU. Effect of dextran concentration on the ultra­ sonic depolymerization...... 89 11# Comparison between acid and ultrasonic degra­ dation on a high molecular weight dextran .... 97 12. Comparison of integral distribution curves for subfractions of degraded dextran B-512 result­ ing from different methods of depolymerization lu3 13• Comparison of differential distribution curves of degraded dextran B-512, resulting from different methods of depolymerization...... luij. -Till- TABLE 25

The molecular weight distribution of acid hydrolyzed dextran 683

Sub­ Sub- * * frac­ % * Wt.-ave. frac­ % Wt.-ave. IPA Fraction tion yield W mol.wt. Fraction tion yield mol.wt...... 32.5 Pi a 7.88 0.56 1)1)5,300 Pi a 35.0 b 2.65 0.46 300,500 * b ...... —————— 37.5 c 0.65 0.36 181),000 c 3.92 0.22 69,000 4 0.0 d 0.90 0.30 127,800 d 1.57 0.21 63,000 1)2.5 e -- .... e 0.80 0.19 .51,300 1)5.0 f .... ——————— f 1.20 0.18 46,000 5o.o g 1.10 0.25 88,750 g 0.40 0.17 41,000

32.5 a 6.68 287,500 Fk a ...... P?2 o.l#5 35.o b 4.13 0.36 184,000 P b .... — 1— 37.5 c 2.30 0.31 136,500 c -... — - -- 1)0.0 d 0.50 0.21*. 82,000 d 4.64 0.19 51,300 1)2.5 e -- .... m* — — — m — tm e 0.30 0.17 41,000 1)5.0 f 0.35 0.21 63,000 f -- — 5o.o g 0.30 0.19 51,300 g 1.52 0.11 17*200 ...... -- -- ...... 32.5 PV a U a 35.0 b .... b ...... 37.5 c 8.32 0.28 111,300 c ...... 1)0.0 d 2.814. 0.23 75,000 d 1.42 0.18 46,000 1)2.5 e -- - — — e 1.92 0.15 32,000 1)5.0 f .... ------f 4.80 0.14 28,000 So.o g 4.82 0,17 1*1,000 g 2.23 0.10 14,200

^'Percentage yield based on original weight ^"^eight-average molecular weight as of dextran before fractionation. measured by light-scattering. -:hhc-jjo fraction was recovered at this alcohol concentration. 8 2

TABLE 26

Effect of ultrasonic vibrations on dextran

Total period Energy* / ■5HS* of output ^ sp/c irradiation watts

0 min. --- 5 .8 8

30 !t 550 3 .3 0 60 it 550 2 .5 0 90 it 550 2.30 120 it 550 2.00 150 it 550 1 .2 0 180 it 550 0 .8 9 210 it 550 0 .6 9 2^0 11 550 0.67

Calculated energy- output, 550 watts = (voltage input) x (current input) 2.20 k.v. 250 m. amps. specific viscosity = Relative viscosity - 1. cosity was found to be 0.67. Preliminary fractionation was performed on 500 ml of a 2 per cent aqueous solution of the degraded polymer. The yields of the fractions were calcu­ lated and their intrinsic viscosities determined. This ex­ periment was repeated and the average results are tabulated in table 27? which show: (1) High molecular weight dextran was depolymerized by ultrasonic vibrations using a calculated energy output of 550 watts. (2) The preliminary test for the molecular weight homogeneity revealed that a total of 83*6 per cent of the degraded polymer has an intrinsic viscosity value of al­ most the same as that before fractionation. (3) The intrinsic viscosity of the degraded dextran was not low enough to reach the desired value of a plasma extender. Therefore, the following experiments were conducted to investigate certain factors affecting the ultrasonic depo­ lymerization.

Effect of dextran concentration. Various concentrations of aqueous solutions of dextran (1, 2, Lj. and 6 per cent) were subjected to ultrasonic vibrations using a calculated energy output of 14.30 watts. Depolymerization was followed by vis­ cosity measurements. The results are given in table 28 and figure 9 , from which the following conclusions may be drawn. SUI­

TABLE 27

Preliminary test for molecular weight homogeneity of an ultrasonic degraded dextran"'

% Weight of % * * IPA Fractions yield f t l in g.

35 7.35 73.5

k o 1.01 10.1 0.63

0.314- 3-14- 0.56 5o 0.10 1.0 O.Ij.9 5 5 No recovery

500 ml aqueous solution of 2 % degraded dex­ tran was subjected to fractional precipita­ tion. The^^value of fractionated polymer was O.67. Percentage yield based on the original ma­ terial for fractionation. 8 5

TABLE 28

Effect of dextran concentration on ultrasonic depolymerization

Total period Energy Specific viscosity/concentration of output of irradiation watts 1 % 2% 6 %

0 min. 1+30 1.87 2.92 5.20 5.88 30 11 1+30 0.87 1.66 3.20 3.1+0 60 11 1+30 0.71+ 1.11+ 2.06 2.50 90 n i+30 0.60 0.91+ 1.50 2.20 120 11 1+30 0.1+3 O .87 1.10 1.90 150 11 1+30 0.38 0.72 0.90 1 .I+0

180 11 1+30 0.30 0.51+ 0.70 1.20 210 11 1+30 0.26 0.50 0.55 0.95 Z 0 h &

OS - GC O'

0 4 -

30 60 9.0 1ZO Time, of irradiation* in minutes 87 (1) There is a direct relation between the rate of* degradation and the percentage of the polymer in the aqueous solution* High concentration calls for longer period of ir­ radiation and vice versa. (2) It seems evident that to reach the desired value of a plasma expander a higher energy output is needed for the depolymerization, especially towards the end of the degradation.

Effect of the molecular weight of dextran. The pre­ vious experiment showed that the ultrasonic treatment of dextran leads to a smooth drop in viscosity, especially in the first 60-90 minutes, which seems to be independent, to a certain extent, of the concentration. This smooth drop is followed by a slow decrease in the degradation process. The following experiment was conducted to explain this ob­ servation. Two samples of dextran, a and b, of different molecular weights were degraded by ultrasonic vibration. Sample "a" has an intrinsic viscosity value of 1.87, while sample I!b" has a value of 0.!{.;>, indicating a high and low molecular polymer, respectively. A 1 per cent aqueous solution of each sample was subjected to the same treatment, using a calculated energy output of Lj_30 watts. Depolymerization was followed by viscosity measurements and the results are given on table 29 and figure 10, from which the following 88

TABLE 29

Effect of tiie molecular weight of dextran on the ultrasonic degradation

Total period Energy Specific viscosity/concentration of • output x, _\ j. ■>__ irradiation watts H. mol. wt.“ L. mol. wt.*'*'

0 min. -- 1.87 0.1+5 30 11 1+30 0.89 0.1+3 60 " 1+30 0.71+ 0.39 90 " 1+30 0.59 0.35 120 " 1+30 0.1+5 0.30 150 " 1+30 0.38 0.27

H.mol.wt. = High molecular weight dextran (sample a). ‘',r"'I.mol.wt. = Low molecular weight dextran (sample b) . F/'fure fO. Effect of dextran. concentration on the ultrasonic depolymerization.

So

4.0 O—

• — 4fo

— o — z i *

— • — !°fo

6 0 /zo Z/O Time of irradiation in minutes 90 conclusions were procured: (1 ) The high molecular weight dextran was degraded smoothly.

(2) The low molecular weight polymer resisted the de­ polymerization process, to such an extent that It can be stated that the ultrasonic treatment acts on the large mole­ cules of the long chains of dextran sooner than that on the short chains.

Effect of calculated energy output. In the previous experiments, by use of ultrasonic vibrations for the degra­ dation of dextran, it was found that the percentage of the polymer and its molecular weight are important factors in the depolymerization. However, it was noticed that under these conditions, a longer period of exposure was required to reach the desired molecular size. This experiment was carried out to study the effect of the calculated energy output (intensity) of the ultrasonic vibrations on the de­ polymerization of dextran.

A 2 per cent aqueous solution of high molecular weight dextran, divided into two parts, was subjected to ultrasonic vibrations.. One part was irradiated, using energy output of J4.3O. watts for a period of 210 minutes. The other part was treated In the same manner, with the exception that an energy output of 630 watts was used. Degradation was fol­ lowed by viscosity measurements. The results, ;Ihich show