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University Micrdfilrns International 300 N Z&eb Road Ann Arbor. Ml 48106 I 8300293
Kwon, Steven Soon*Young
PHYSICAL AND CHEMICAL PROPERTIES OF CARRAGEENAN FRACTIONS OBTAINED BY SEQUENTIAL EXTRACTION OF CHONDRUS CRISPUS
The Ohio State University PH.D. 1982
University Microfilms International300 N. Zceb Road, Ann Attoor, MI 48106
Copyright 1983 by Kwon, Steven Soon-Young All Rights Reserved
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University Microfilms International
PHYSICAL AND CHEMICAL PROPERTIES OF CARRAGEENAN
FRACTIONS OBTAINED BY SEQUENTIAL EXTRACTION OF
CHONDRUS CRISPUS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the
Degree Doctor of Philosophy ’in the Graduate School of
The Ohio State University
By
Steven Soon-Young Kwon, M.S.
The Ohio State University
1982
Reading Committee: Approved by
P. M. T. Hansen
T. Kristoffersen
M. E. Mangino Department of/Food Science J . B . Lindamood and Nutrition ACKNOWLEDGEMENTS
I am sincerely grateful to Dr* Poul M. T. Hansen for his support and guidance during the entire period of my study at OSU. His vital interest in research and affection toward his students will be a valuable lesson in my future career.
1 am extremely appreciative of Dr. T. Kristoffersen for his support in my academic career.
I am also grateful to Drs. M. E. Mangino and J. B.
Lindamood for their suggestions and careful reading of my dissertation. VITA
December 12, 1947 Born: Chon-Ahn, Korea
February, 1972 B. Agr. in Agricultural Chemistry, Korea University Korea
1972-1973 Mew Zealand Government Scholarship Trainee in Dairy Technology, Palmerston North, N.Z.
1973-1976 Section Head, Processing and Quality Control, Aseptic Fluid Milk Division, Korea Dairy Company
1979 M.S. (Food Science), University of California at Davis
1979-1982 Research Associate, Department of Food Science & Nutrition, The Ohio State University
PUBLICATIONS
1. Kwon, S., R. A. Bernhard and T. A. Nickerson. 1981. Recovery of lactose from aqueous solutions: Precipitation with magnesium chlorides and sodium hydroxide. J. of Dairy Sci. 64(3):398-406.
2. Kwon, S. Y. and T. A. Nickerson. 1978. Lactose recovery with magnesium chloride. J. of Dairy Sci. Supplement 1. 61:112. Abstract.
iii TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS...... ii
VITA ...... iii
LIST OF FIGURES...... vi
LIST OF TABLES...... viii INTRODUCTION...... 1 LITERATURE REVIEW...... 3 A. Extraction of Carrageenan from Red Marine A lg a e ...... 1. The Heterogeneity of Carrageenan...... 3 2. The Physical Structure of Carrageenan in the Native State...... 11 3. Carrageenan Gel as Related to Carrageenan Extractability from Chondrus crispus..... 23 4 .Methods of Carrageenan Extraction ......
B. The Stabilization of Calcium Sensitive Milk Proteins by Carrageenan...... 43
C. Molecular Weight and Size of Carrageenan.... 54 D. Molecular Weight Determination by Analytical Ultracentrifugation...... 62
MATERIAL AND METHODS...... 67
A. Carrageenan Sample...... 67 B. Sequential Extraction of Carrageenan from Chondrus crispus...... 67
C. Gel Filtration...... 70
D. Analytical Ultracentrifugation...... 71 £. Viscosity Measurement...... 71
F. Refractive Index Determination...... 72 iv G. Zonal Electrophoresis...... 74
H. KC1 Fractionation o£ Carrageenan Extracts 74
I. Stabilization of Alpha (Si) -Casein...... 76
RESULTS...... 79
A* Sequential Extraction of Carrageenan from Chondrus crispus...... 79 1. The Extraction Yields of Carrageenan ...... 79 2. KC1 Fractionation of Kappa-, lota-, and Lambda-carrageenan from the Sequential Extracts...... 8 0 3. Zonal Electrophoresis...... 91
B. Molecular Weight and Size...... 94 1. Gel permeation Chromatography...... 94 2. Molecular Weight Determination by Analytical Ultracentrifugation...... 100 3. Intrinsic Viscosity...... 105 4. Refractive Index Determination...... 108
C. Stabilization if Alpha (SI)-Casein...... n o
DISCUSSION...... 117
A. The Mode of Sequential Carrageenan Extraction at Gradually Elevated Temperatures...... 119
B. The Physical and Functional Properties of the Sequential Extracts......
C. An Effective Method of Carrageenan Extraction
SUMMARY...... 124
REFERENCES...... 128
APPENDIXES...... 143
A. Molecular Weight Determination by Gel Permeation Chromatography...... 143
B. Molecular Weight Determination by Sedimentation Equilibrium...... 145
v LIST OF FIGURES
Figure PAGE
1 . Repeating sugar units of carrageenans ......
2 . Ultrastructure of young cells of Chondrus crispus... 13
3. Ultrastructure of Chondrus crispus carposporic cells. 17
Ultrastructure of Chondrus crispus after extraction.. 4. 18 5. Possible mode of aggregation of kappa-carrageenan molecules in solution with hydrated univalent cations • * * 2 0
6 . Proposed arrangement of polysaccharide chains in iota • • *22 7. Gelling mehcanism of carrageenan*.*...... 26
8 . Schematic model for a two-step gelation mechanism in carrageenan...... 28
9. The domain model of carrageenan gelation...... 29
10. The interaction of kappa-carrageenan with alpha(SI)- casein in the presence of calcium...... 47
11. The sol-gel transition as proposed for kappa-and iota carrageenan in milk.*...... 51
12. Experimental outline of physical chemical and functional characterization of carrageenan fractions of Chondrus crispus...... 68
13. KC1 precipitation curve of carrageenan extraction samples...... 87
14. Concentration of kappa-, iota-, and lambda-carrageenan in the extraction samples of Chondurs crispus...... gg
15. Zonal eletrophoretic patterns...... 93
vi 16. Gel permeation chromatogram o£ the 40C extract .%
17. Gel permeation chromatogram of the 70C extract..9 7
18. Gel permeation chromatogram of the HOC extract..93
19. Intrinsic viscosity ...... 106
vii LIST OP TABLES
Table PAGE
1 Major families of red marine algae, Rhodophyceae; Source of carrageenan...... 4
2. Energy-dispersive X-ray analysis (EDX) of carposporic and tetrasporic cells in sulphur counts per minute...... ^5
3. Preparation and properties of carrageenan fractions from Chondrus crispus...... 37
4. Molecular weight and size properties of carrageenan...... 55
5. Centrifugal analysis of carrageenan...... 53
6 . The yields of carrageenan extracts sequentially obtained from Chondrus crispus at gradually increasing temperatures...... 81
7. Precipitation (%) of the carrageenan extraction fractions at varying potassium chloride concentrations...... 83
B. Analysis of variance; Precipitation of carrageenan * extracts by potassium chloride...... £A
9. Duncan's multiple range test: precipitation of carrageenan extraction samples by potassium chloride...... 3 5
10. Fractional composition of kappa-, iota-, and lambda-carrageenan in the extraction samples obtained from Chondrus crispus...... gg 11. Fractional yield of kappa and lambda as per cent of the total weight of the carrageenan extracts...... 12. The molecular weights and molecular size of the carrageenan extraction samples...... 99 13. Apparent weight average molecular weights, Mw(app), of the carrageenan extraction samples at three different speeds and at three different concentrations...... JD2 viii 14. Corrected true molecular weight, Mw(corr), and best fit multiple regression model...... 2,04 15. Intrinsic viscosity of the carrageenan extract and corresponding Mw by GPC and Mw(app) by sedimentation equilibrium...... 1 0 7 16. Refractive index of the carrageenan extract in solid state and liquid state...... io9 17. Stabilization of alpha(Si)-casein by the carrageenan extraction samples...... 18. Analysis of variance: stabilization of alpha(Sl)-casein by the carrageenan extract samples...... 2 1 2 19. Duncan’s multiple range test: stabilization of alpha(Sl)-casein by the carrageenan extract samples. . . . 1 1 3 20. Correlation between physical, chemical parameters, and casein stabilization...... 125 21. The effect of molecular inhomogeneity on the milk protein stabilization...... 115 xx INTRODUCTION Carrageenan has attained importance in the food industry as a stabilizing) thickening and gelling agent. It possesses the ability to stabilize calcium sensitive protein, notably alpha(SI)-casein, a function which may be significant in the stabilization of evaporated milk and infant formula. A major source of carrageenan includes a variety of species of the red algal seaweed, Rhodophyta. The functionality of carrageenan as a stabilizing agent depends critically upon the methods used for extraction from the seaweed* A common method for commercial extraction involves a one-time cooking process of the seaweeds at or near boiling temperature in slightly alkaline aqueous media. The carrageenan extract obtained is thus a mixture of different carrageenan varieties including highly functional as well as non-functional fractions. The current study was undertaken to improve the understanding of the sequence mode of carrageenan extraction from Chondrus crispus with the underlying purpose of designing an extraction method which may selectively produce carrageenan fractions for specific uses. 1 2 The current investigation showed tK at carrageenan fractions which are distinctively differc nt in their chemical, physical, and functional pro;Gerties can be obtained by sequential extraction of the plari t material at gradually increasing temperature* LITERATURE REVIEW A. Extraction of Carrageenan from Red Marine Algae Certain red seaweeds give rise to a group of natural phycocolloids (mucilages and gums) with a great variety of uses in food preparation. While the cell walls of most plants contain a high concentration of cellulose, the cell walls of marine algae are composed of other polysaccharides and less than 11 of the dry weight of the plant is cellulosic (Young, 1966). Carrageenan is the name applied to the mixture of the water extractable polysaccharides obtained from the cell walls and intercelular matrices of the red marine algae. 1. Heterogeneity of Carrageenan The major families of the red marine algae, Rhodophyceae, and its botanical descendants are shown in Table 1. The principal seaweed as a source of carrageenan is Chondrus crispus, also known as Irish moss, though in some places Gigartina stellata (Gigartlna mamillosa) is also employed. Studies (Taylor and Chen, 1973; Chen and McLachlan, 1972) on the life history of Chondrus crispus have shown that three different genetic forms occur within the same 4 Table 1. Major Families of Red Marine Algae, Rhodophycea: Source of Carrageenan. Family Genus Species Carrageenan Furcellariaceae Furcellaria F.fastigiata Kappa Solieraceae Agardhiella A.tenera lota Eucheuma E.spinosum Iota E.cottonii Kappa,lambda • Anatheca A.montagnei Iota Hypneaccae Hypnea H.musciformis Kappa H.nidifica (Hawaii) Kappa H.setosa (Hawaii) Kappa Gigartinaceae Chondrus C.crispus Kappa,lambda ( iota C.sp.(Hawaaii) Lambda * Gigartina G.stellata Lambda,kappa, iota G.acicularis Lambda,kappa G.piBtillata Lambda,kappa Iridaea I.radula Iridophycan, kappa,lambda Phyllophoraceae Phyllophora P.nervosa Phyllophoran Gymnogongrus G.sp*(Hawaii) lota (Adapted from Chapman and Chapman, 1900). I 5 species * The male plant and the female plant (the gametophytes) produce reproductive cells (gametes) which fuse, and from spores that develop as a result of this fusion, an asexual plant (the tetrasporophyte) arises. Tetraspores from this plant result in second generation gametophytes. Gametophytes derived from the tetraspores become sexually mature within a year(Chen and McLachlan, 1972) It is now known that carrageenan is chemically a group of galactan polysaccharides extracted from seaweeds of the Gigartinaceae, Solieraceae, Hypneaceae and Phyllophoraceae families that have an ester sulfate content of 18% or more and are alternately alpha-1,3; beta-1,4 glycosidically linked. Even though the use of carrageenan can be traced back at least two centuries (Guiseley, 1968), very little was known about its chemical structure until the mid-1950's. Smith and Cook (1953) showed that carrageenan extracted from Chondrus crispus can be separated into two fractions, kappa- and lambda-carrageenan, differing in chemical composition and sulfate content. In this case, separation is achieved by precipitation of the kappa-carrageenan from a 0 .2 % aqueous solution with 0.125-0.25M KC1 solution; the fraction remaining in solution is lambda-carrageenan. Later the chemical structure studies (O'Neil, 1955; Rees, 1969B) on the two fractions from the ChondruB crispus revealed that lambda-carrageeenan consists of 1 , 4 linked galactose-2,6 -disulfate and 1,3 linked galactose-2-sulfate units leading to a sulfate content of about 37%, while kappa-carrageenan consists of 1,3 linked galactose-4-sulfate and 1,4-linked 3,6-anhydrogalactose units (Fig. 1) with a sulfate content of abuut 23%. A third carrageenan component, iota-carrageenan, has been extracted mainly from the Eucheuma species, for example, Eucheuma spinosum. The chemical structure of iota-carrageenan (Mueller and Rees, 1968) is identical with kappa-carrageenan except for the sulfate group on the C2 position of the anhydrogalactose unit (Fig. 1). In addition to the three major types of carrageenan, kappa-, lambda-, and iota-carrageenan, other forms of carrageenan have been separated and their structural patterns investigated (Anderson et al, 1968; Standoff and Stanley, 1967; Rees, 1969B; Yaphe, 1973). Highly theoretical structures of the heterogeneous carrageenans, mu, kappa, nu, iota, lambda, and xi, have been proposed (Mueller and Rees, 1968, Stanley, 1970). The fractions may be interrelated with one another aB precursors or as metabolism end products to one another. As shown by Fig. 1, kappa-carrageenan is derived from mu, iota from nu, and theta from lambda. The heterogeneity of carrageenan prevailing in the red algal species of Rhodophyta did not receive much attention 7 — V/T—*'..— *A'*— ■ * B A CH,OH CHjOtO,- CM,OM CH, OH DM OH OH «U W KATfA ( |) CH/7H 04,0*0,- CM CM, OH 0*0,- OH 010/- H U (•) IOTA(,) CH/JH, CM jOSO j- CMyOH CH, jd~~ y^yTV o^. pr°~>(V0|X0^ I to,-< 70%1 °4<^ (VV-fTOS) M0*“ tAHtU «* THtTA («) Pig i. Repeating units of carrageenans. until the structural studies on the carrageenan fractions were reported by Rees and co-workerB in the 1960's, since then various factors have been reported which are responsible for the heterogeneous nature of carageenan in the plants (Black et al., 1965; Parsons et al., 1977; Chen et al., 1979). It is a well accepted fact that the composition of carrageenan varies widely depending on the habitat of the species both within and outside the same genera (Black et al., 1965). In addition to interspecies heterogeneity, the constituents vary even with the same species. The factors responsible for the variability include geographic location (Mathieson and Tveter, 1975), seasonal variation (Dawes, 1978; Dawes et al., 1977), other ecological factors (Mathieson and Tveter, 1975), and most importantly the reproductive stage in the life cycle (Chen et al.,1973; Pickmere et al., 1973, McCandless et al., 1975). The content and fractional composition of carrageenan in red algal seaweed appears to be dependent on the variation in the geographic location; the estuarine population of Gigartina (Mathieson and Tveter, 1976) exhibited a greater carrageenan content than the open coastal populations while Chondrus crispus showed the opposite trend. Spatial variation, whether the plants grow in intertidal or subtidal zones, affects the total concentration o£ carrageenan in the plants; subtidal populations o£ ChondruB crispus showed the highest content o£ carrageenan (Mathieson and Tveter, 1975). In their ecological studies o£ Chondrus crispus, Mathieson et al. (1973 and 1975) suggested that the seasonal variation greatly affected the percentage of carrageenan with maximum values in the autumn and early winter. The total yield of carrageenan from Floridian Eucheuma (Dawes, 1978) also showed cyclic changes with the season, although the molar ratios between kappa and lambda fractions were unchanged throughout the year. The effect of the seasonal variation on the carrageenan content is not always apparent; Pickmere et al. (1975) found no significant variation in the level of carrageenan in the three species of Gigartina. It appeared that the seasonal biochemical changes in the algal plants are the complex interaction products of many factors such as temperature (Chen et al., 1979), salinity, nutrients, and intertidal exposure (Mathieson and Tveter, 1975) So far the most convincing evidence which can account for the variability of carrageenan fractions within the same species of Chondrus crispus is the reproduction stage in the life cycle of the habitat plant (Chen et al., 1973); Pickmere et al., 1973; Waaland, 1975). According to the study of Chen and McLachlan (1971), 10 A life cycle of Chondrus crispus takes about 20 months in culture. Tetraspores of plants form male and femal gametophytes and subequently carposporophytes; carpospores in turn give rise to plants with mature tetrasporangia. Tetraspores from these result in second generation gametophytes. The quantitative ratios of kappa-to lambda-carrageenan in Chrondus crispus (Chen et al., 1973) is low in tetrasporic plants and relative high in gametophytes. Pickmere et al. (1973) reported in their study with Gigartina species that the total carrageenan level showed very little variation between male, female, and tetrasporic plants; however, the male and female gametophyte plants exhibited a higher kappa to lambda ratio than the tetrasporophytes. A similar pattern was also observed in other generic species of the Gigartinaceae and Iridaea families(McCandless et al., 1975; Waaland, 1975). In the gametophyte of Irideae cordata , 93% of total carrageenan was of the kappa fraction; in tetrasporophyte, 95% was lambda-carrageenan (Waaland, 1975). Hamic (1973) has suggested that the seaweed industry may selectively cultivate the commercially important seaweed species of Chondrus, Gigartina, and Irideae to improve the content of the plants. It was suggested that the vegetative propagation of the gametophyte phase (Taylor and Chon, 1973) can be achieved in large quantities, if industry needs to 11 produce preferentially the gel-forming kappa-carrageenan. If so, then the site of propagation would be an important consideration because the growth of the sexual plants appears to be related to geographical location. For example, 90% of Chondrus crlspus collected at Sandy (Fink) Cove was gametophytic (Taylor and Chen, 1973). 2. Physical Structure of Carraqeenan in the Native State In general, the cell walls of most plants are composite materials and contain basically a fibrillar component along with a mucilaginous substance. The mucilage is usually regarded as forming a non- or para-crystalline matrix in which the microfibrils are embedded. The microfibrils form the most inert and resistant part of the cell wall (Mackie and Preston, 1974). The most common of these skeletal components in plants is the neutral polysaccharides, cellulose. However, the cell walls of red marine algae, especially Choudrus crispus and related species, contain very little cellulose; less than 1 % of the total dry weight of the plant (Young, 1966). Instead, the cells of the red algae are, in general, coated with a gel state anionic 12 polysaccharide, carrageenan, (Ramus, 1973; Kloareg, 1981). Depending on the degree of cross-linking between the polysaccharide polymers as well as the extent of the sulfation of the constituent polysaccharides, the conformation of the interconnected network varies from crystal lattices, to fibres, to completely amorphous structures (Ramus, 1973). Several workers (Taylor and Chen, 1973; Gordon and McCandless, 1973; LaClaire and Dawes, 1976) have investigated the ultrastructure of the cells of the red algal plants and suggested that the sulfated polygalactans, carrageenan, are the major elements of the microfibrils of the cell wall as well as the intercellular matrix. The cell walls are layered and consist of microfibrils arranged circumferentially but random in surface views (Fig. 2) while the intercellular matrix appears more granular than the cell walls. In their study on the localization of carrageenan in Chondrus crispus, Gordon and McCandless (1973) suggested that kappa-carrageenan occurs in the plant as crystalline cell wall microfibrils, and lambda- and mu-carrageenans occur as an amorphous intercellular and perhaps interfibrillar matrix. A consistent result supporting this view may be found in the study by McCandless et al.(1977) who showed that the amount of sulfur increased with the 13 Fig 2. Ultrastracture of section of young cells of Chondrus crispus showing golgi, endoplasmic, reticulum , nucleus, vesicles in the cytoplasm, as well as circumferentially arranged microfibrils of cell wall. G: Golgi body. HR: endoplasmic reticulum. N: nucleus. V: vesicle. MG: microfibrils. IM: intercellular matrix. (From Gordon and McCandless, 1973) 14 analysis proceeding from the innermost region of the cell wall to the intercellular matrix (Table 2). The ultrastructures of different genetic types of Chondrus crispus, carposporophyte and tetrasporophyte, appeared to be similar with respect to the concentration of microfibrils which increase from the inner to the outer region of the wall (McCandless et al., 1977). Eucheuma nudum, a species rich in iota-carrageenan, was studied by LaClaire and Dawes(1976) who used an autoradiographic and histochemical method. They suggested that the predominant sulfated polysaccharide, iota-carrageenan (Fig. 1), was localized in the middle lamella of the epidermal, cortical and medullary cells of Eucheuma nudum. Ultrastructually the Eucheuma plant closely resembles Chondrus crispus? however, the quantity of the wall material relative to cytoplasm was strikingly low. Unlike Chondrus crispus, the plant appeared to contain some cellulose located in the inner portion of cell walls in addition to iota-carrageenan. Based on the autoradiographic study with the plant cells, they proposed that the backbone polysaccharide (non-sulfated polygalactan) is produced by the golgi apparatus and extracellular sulfation takes place by the sulfotransferase (Craigie and Wong, 1978) secreted by endoplasmic reticulum. Table 2. Energy-dispersive X-ray (EDX) analysis of carposporic and tetrasporic cells in sulphur counts per minute > Genetic Area Inner Outer Intercellular type______plant cell wall cell wall______matrix 1 132 166 Tetraspo Stipe . 37 125 111 ric cells 59 298 388 Frond 17 148 574 251 1265 892 Carpospo Stipe 288 1136 1154 ric cells 133 1118 442 Frond 313 758 519 t (Adapted from McCandless et al.,.1977)» 1 tn 16 From an industrial view point, the localization of the carrageenan fractions in Chondrus crispus and other related species provides useful information which relates to the mechanism and conditions of extraction on a commercial scale. McCandless et al. (1977) studied the localization of carrageenan in Chondrus crispus, and obtained information with respect to the effect of the extraction of carrageenan on the cellular structure of Chondrus crispus. The ultrastructure and the birefringence of cell walls and intercellular matrix(Fig. 3) were observed before and after the extraction. After two hours of extraction at 95C in a sodium bicarbonate buffer solution, the microfibrils in the cell wall appeared to have been reduced in concentration and compacted at the wall-matrix interface (Fig. 4). The net work of microfibrils (presumably kappa-carrageenan in the gel state) had been disrupted while the intercellular granules (presumably lambda-carrageenan) was significantly reduced. Birefringence of the cell wall was greately reduced by a 2 hr extraction, and intercellular matrix birefringence was entirely removed. From this, the workers suggested that the outer edge of the cell wall resisted extraction better than the rest of the cell. The ultrastructural change due to the extraction has not been reported at the molecular or atomic level. Rees 17 i vtyst Fig 3. Ultrastructure of Chondus crispus carposporic tip cells. The microfibrils (MB) in the cell wall are concentrically arraged and are much less dense in the inner cell wall (ICVO than in the outer cell wall (OCW). The intercellular matrix (IM) is granular in appearance. Cytoplasm (C). Bar represents 0.5um. x 24,300. (From McCandless et al.. 1977} 18 SiPjp>:-ffl.ir:*f .'jV; Fig 4. Ultrastructure of Chondrus crispus carposporic frond cell which bad been extracted for 2hr(A) and 5hr(B) in hot NaHCOg. Bar represents 0.5um. x 37800. (from McCandless et al., 1977) 19 (1981), however, suggested from his study with cation-mediated carrageenan gels that the physical rupture of the aggregated carrageenan gel (which simulates the carrageenan state in vivo) does not require the breakage of covalent bonds. The physical structure of various carrageenan gels have been studied at the atomic level by the X-ray diffraction method. In 1955, Bailey revealed the molecular structures of kappa- and lambda- carrageenan from his x-ray diffraction study. He reported that the molecular chain of the sodium salt of kappa-carrageenan shows a fully extended form with a 25.2 A fiber period and proposed a molecular packing arrangement illustrated as in Fig. 5. As shown in Fig. 5, two kappa-carrageenan molecules are held closely together by electrostatic forces between their nearest sulfate groups and the univalent cations. Sodium salts of lambda-carrageenan also showed a 25.2 A of fiber repeating distance (Bailey, 1955). The overall shape of lambda-carrageenan resembles a rather flat, extended ribbon bending somewhat from side to side (Rees, 1969). According to the X-ray diffraction photographs later obtained by Anderson et al.(1969A), it was suggested that monovalent cation salts of kappa-and iota-carrageenan have fiber axes repeat distance of 24.6 A and 13.0 A respectively. The oriented fibre salts (Fig. 6) showed double helices with 20 Fig 5. Possible mode of aggregation of kappa-carrageenan molecules in solution with hydrated univalent cations. (From Baileyi 1955) 21 three disaccharide residues in a complete turn of each single chain in 24.6 A (kappa) or 26.A (iota). In iota-carrageenan the second chain is displaced exactly half a pitch from the first as shown in Fig. 6. Both kappa-and iota-carrageenan con forma tiojns are stabilized by specific-hydrogen bonding between 0(2) and 0 (6) of galactose residues in different strands of «L.the same double helix (Anderson et al.,1969A). The molecular structure of iota-carrageenan was further investigated by Arnott et al. (1974). The fibre diffraction patterns| of the highly crysptalline Ca++, Sr++, and Mg++ salts of iota-carrageenan, which they examined, showed similarity with the model reported by Anderson et al. (1969) The molecular model consists of two identical, right L handed, 3-fold helical polysaccharide chains of a fiber repeating distance of 26.56 A with one chain translating axially 13.2 A relative to the other. The workers noted a large interhelical distance ( 2.7 A) of the divalent cation salts of iota-carrageenan and proposed that the ionic interaction is more important in holding the crystal structure together than the hydrogen bonding between the double helix or the Van der Waals forces. Each cation is coordinated to two sulfate groups, each from a different helix in such a way as to produce a - Ca-sulfate-Ca-sulfate-.... chain punning through the lattice parallel with the fibre axis. 22 ••i e < np rCc^sO^------ ) ' * 120 3«f 4 Fig 6. The proposed arragement of polysaccharide chains in iota. Two chemically distinct chains, represented by full and broken lines, respectively, and each having three disaccharide residues in one turn of helix of pitch 26.0 A, are twisted together to form a double helix with a translation period of 13.0 A. The structure is represented as a projection on the surface of a cylinder which has then been opened out. Small circles represent oxygen atoms and large circles are S03- groups. (From Anderson et al., 1969) 23 Under the polarizing light microscope only crystalline or highly ordered substances exhibit birefringence (West,1954; McCrone et al., 1978). Whether or not kappa and iota carrageenanB in situ exist only as the cation-salt forms is not fully known, but it is possible that the birefringence of the cellular matrix of Chondrus crispus, observed by McCandless and other workers, could in part be contributed by these highly ordered cation-salts of the carrageenans. 3.Carrageenan Gel b b Related to Carrageenan Extractabllity from Chondrus crispus The hot-water extraction of carrageenan from the plant tissue of Chondrus crispus results in the disintegration of both the microfibrils and the intracellular matrix. The degree of cellular decomposition and the physical and chemical properties of the polysaccharide can vary widely depending on the extraction method employed (McCandless et al., 1977). It is generally accepted that the microfibrils of the plant cell wall are made of a gel state kappa-carrageenan (Gordon and McCandless, 1973; Chapman and Chapman, 1980). As reported by Gordon-Mills and McCandless (1977), the 24 microfibrils observed by an electronmicroscope exhibit a similar structural orientation as the kappa-carrageenan gel in vitro seen under a polarizing microscope. These observations indicate that the extraction mode of kappa-carrageenan from the microfibrils may be related to the behavior of the kappa-carrageenan gel ^n vitro. The gelation of carrageenan extracts is dictated by the molecular structure of the component carrageenan. As shown by Rees (1969). the structure of kappa- and iota-carageenan permits the formation a gel which is not possible for lambda- carrageenan. The gelation of carrageenan is also affected by the temperature {Stanley, 1970; Reid et al., 1974), the presence of counterions (Payens and Snoeren, 1972; Rochas and Rinaudo, 1980; Rees, 1981), and concentration (Bryce et al, 1982). As suggested by Rees and his coworkers (Rees et al., 1969; Morris and Benton, 1980), a thermally reversible gel can be formed from a diluted aqueous solution of kappa- and iota-carrageenan. The gelation of carrageenan in the presence of the counter-ions has been proposed by several workers, most notably by Rees and his coworkers, and Smidsrod and his colleagues (Smidsrod, 1980). Their models of carrageenan gelation agree with each other in the 25 principle that gelation requires a conformational change of carrageenan from a random coil to an ordered state which then undergoes further association to form a gel network in the presence of gel promoting cations. The basic difference between the two models, however, lies in the presence or absence of a helical interaction. Rees and his coworkers (Rees et al., 1981), suggested in their earlier model that the gelation mechanism involves the conformational transition of the carrageenan from a random coil to a double helix followed by intramolecular hydrogen bonding as shown in Fig. 7(Gel I), leading to the formation of a three dimensional network (Gel II) in which the double helices form the "junction points" of the polymer chain via hydrogen bonding. The theory of double helix formation has been supported by the observations made by optical rotation methods (Snoeren , 1976; Morris, 1980), differential scanning calorimetry (Reid et al., 1974; Snoeren, 1976), and X-ray diffraction crystallography (Arnott et al., 1974). However, helix formation as an important feature in gelation of carrageenan was questioned by the Smidsrod and his coworkers (1980). They expressed doublts about the role of the mechanism proposed by Rees (1969) on three accounts: Solution CHI 6*1 II Pig 7. Gelling mechanism of carrageenan. (From Rees, 1963) 27 1). Firstly, double helix formation between different molecules and parts of molecules to form a network structure represents a serious mechanistic problem. 2). Secondly, double-helix formation does not explain the salt specificity of gel-formation. 3). Thirdly, double helices have been postulated to be formed both in kappa-and iota-carrageenan and offer, therefore, no explanation of the markedly different gel strengths of these carrageenans. The Norwegian workers proposed a two step gelation process (Fig. 8). In step one, an intramolecular (not intermolecular) conformational change takes place, which is not ion-specific. In step two, ion-specific gelation of carrageenan takes place following a decrease in solubility due to the formation of ion-selective salt-bridges as suggested by Bailey (1955) in his X-ray study with kappa-carrageenan. The "junction zone" model of Rees and his coworkers was later replaced by a "domain model" (Fig. 9) which still presents, but in much less degree, the idea of double helix formation. In that model, the primary mode of interchain association involves the coil-domain transition during which the small soluble clusters of chains (domains) are formed through the intermolecular association of iota-carrageenan chain in double helices (step 1). In the presence of the gel promoting cations such as K-f, Rb+, Cs-f, NH4+, or high 28 ITEP2 Fig B. Schematic model for a two-step gelation mechansism in carrageenan. (From Smidsrod, 1980) Aggregate m // Domain $ Fig 9* The domain model of carrageenan gelation. (From Rees, 1981) 30 concentrations of Na+, these domains undergo further association to develop an infinite gel network via cation-mediated aggregation of the double helices (step 2). The gelation can also occur by a direct coil-aggregate transition (step 3). Bryce et al. (1982) supported this view by their study with iota-carrageenan* They reported that "the transition was qualitatively concentration-dependent, indicating that the intramolecular cross-linking mechanism is not applicable. In the presence of salts, the concentration effects seem to be still consistent with the mechanism". They found also that at the equilibrium state of the coil/helix transformation in the presence of NaCl, the carrageenan (kappa) double helices model consists of the apparent average number of molecules of 3.7 which indicates that if double helices form they aggregate further (Bryce et al., 1982). In its native state in the plant, kappa-carrageenan is associated with the cations of alkaline or alkaline earth metals (Chapman and Chapman, 1980). Among these the metals, K+, Rb+, Cs+ and NH4+ are known to promote the gelation of kappa-and iota-carrageenan while Na+ and Li+ are ineffective. Ashton et al. (1977) suggested that the binding affinities of the inorganic ions to the carrageenans are the greatest with the smallest hydrated ions and decrease in 31 proportion to the hydrated ion size. In particular kappa-carrageenan forms the strongest gel with Cs+ and K+(Orasdalen and Smidsrod, 1981; Rochas and Rinaudo, 1980; Pass et al. 1978) while iota-carrageenan does so with Ca++ (Morris and Benton, 1980). This ion selectivity (or specificity) of carrageenan gels is also reported elsewhere (Pass et al., 1978; Grinberg et al., 1980; Rinaundo and Rochas, 1981; Morris, 1980; Rees, 1981). In his X-ray diffraction study with the kappa-carrageenan gel, Bailey(1955) cited: The gel promoting cations, K+, Rb+, Cs+ and NH4+ have about the same ionic radius in the hydrated state in such a manner that they form Hthe just right size" to fit into the interstices between the kappa-carrageenan chains and lock them together. This type of counter cations-carrageenan interaction tends to considerably increase the molecular weight of the carrageenan polymer(MorriB et al., 1980). For example, Rees (1981) observed that K+ ion-promoted gel of iota-carrageenan showed six to eight times larger the molecular weight of the starting value. The melting or setting transition behavior of the carrageenan solution is also affected by the presence of salt and/or the ionic strength (Rinaudo and Rochas, 1981). 32 Payena and Snoeren (1972) reported that the setting and melting temperatures of the kappa-carrageenan gel depend linearly on the square root of the electrolyte concentration, while Rinaudo et al. (1979) noted a linear dependence with the logarithm of the ionic strength with iota-carrageenan. A similar observation was later reported by Bryce et al. (1982). In their study with the gelling of kappa-carrageenan by potassium salts, Rinaudo and Rochas (1981) noted* When the gel is heated, first the isolated helical chains melt at a temperature TM1; in a second step, the aggregates of helical segments melt at a temperature TM2 while only one melting temperature around TM1, is obtained when the gel is cooled. The physical property of the carrageenan gel is largely affected by the concentration of the constituting carrageenan. In recent studies, Bryce et al.(1982), and Rochas and Rinaudo(1980) noted the concentration dependence of the order-disorder transition of kappa-carrageenan. Rinaudo and Rochas(1981) observed that the transition temperature increases as the concentration of carrageenan segments increases; the increase was even greater in the presence of counter-ions. 33 4. Methods of Carrageenan Extraction/Fractionation Commercial method of carrageenan manufacture from red seaweed varies from one manufacturer to the other. Carrageenan separation commonly involves the steps of extraction of carrageenan from raw material, purification, and dehydration in the following manner(Review* Kardosh, 1975)* plant washing- plant cooking in alkaline solution at 90C- filtration- bleaching by active carbon treatment- precipitation with methanol or drum drying- washing with methanol and ether. Various extraction conditions have been investigated with selected red seaweeds (Cook et al., 1954; Black et al., 1965; Lin, 1971). It has been known that the manner by which the seaweed has been extracted can dictate to a large extent the properties of the final product such as the products yield, types of carrageenan, (Cook and Smith, 1953; Smith et al., 1954), physical and functional properties, including molecular weight (Black et al., 1965; Tong et al., 1980 and 1981), molecular size (Goring and Young, 1954; Renoll and Hansen, 1979), and milk protein reactivity (Lin, 1971; Kardosh, 1975). The major difference between the commercial methods may lie in the stage of plant cooking. For instance, the use of alkaline media instead of water only promotes the conversion of lambda-carrageenan to a more functional theta-carrageenan with respect to gelling and 34 milk protein reactivity ( Stanley, 1963). The major variables commonly employed involve; 1) the chemical nature of the extraction media with respect to type of salt, ionic strength, and pH, and 2) the extraction temperature(Stancioff, 1965) a) Variations in the chemical nature of the extraction media; Alkali (la) and alkaline earth metals (Ha), when present as counter ions, will induce gel formation of carrageenan. The cations of Rb+, Cs+, K+, Ca++, Mg++ are known to promote gel formation while the larger cations (in hydrated form), Na+ and Li-f, do not (Bailey 1955). Kappa-carrageenan forms the strongest gel with K+ ion while iota-carrageenan does so with Ca++ ion. Due to this counter-ion specificity, the fractionation of carrageenans into kappa-, lambda-, or iota- fraction depends largely on their gel forming ability or precipitability with the cations, especially K+ and Ca++. Therefore, the fractionation of carrageenan from the seaweed plant tissues is commonly preceded by an extraction step using either water as the extraction solvent (Tong et al., 1980 and 1981) or solutions of non-gelling salts such as dilute NaCl (Black et al., 1965) or dilute solutions of NaCl and Na2C03 (Smith et al., 1955) in order to maximally solubilize the 35 carrageenan in the plant without causing gelation* Cook and Smith (1954) separated carrageenans according to their solubility in 0.25M KC1 solution. The precipitated fraction was named kappa-carrageenan and the soluble fraction* lamda-carrageenan. The KC1 precipitation method of Smith and Cook (1953) was then applied to the various species of Chondrus* Oigartina* Euchenma* and Polyides by Black et al.(1965). From their structural analysis of the plant extracts* they noticed that the two fractions* KC1 soluble and precipitate, varied considerably in 3*6 anhydro-galactose(3,6 AO) and sulfate contents from one r species to another as well as among the different samples from the same species. The fractionation method of Cook and Smith (1953) was improved to obtain and characterize the carrageenan fractions in more sensitive manner(Smith et al., 1954; Pernas et al.* 1967; Matulewicz and Cerezo, 1980). Smith et al.* (1954) added different concentrations of potassium chloride successively from 0.05 to 0.275M into the aqueous carrageenan solution of Chondrus crispus at 25C and noticed that most of the kappa fraction precipitated up to 0.15M KC1. Pernas et al.(1967) employed an even broader range of KC1 concentrations from 0.0625M to 1.5M in order to fractionate the initial carrageenan extracts obtained from Chondrus criBpus* Gigartina Btellata, and Gigartina 36 skottsbergll. As shown by Table 3f wide variations in yield, chpraical composition, and gel strength were noted between the fractions. From the study they concluded that The carrageenans cannot consist of a mixture of only two components, kappa and lambda, according to the classical definition, but rather a series of molecules of different chemical composition and consequently, with different solubility. A similar view was pointed out later by Matulewicz and Cerezo (1980) who scrutinized the KC1 precipitation method in their study with Iridaea undulosa B.. The workers subdivided the initial carrageenan extracts from the plant into eight fractions according to their precipitability with KC1 from 0.125M to 2.0M. They regrouped the fractions into the three major components which are distinctively different from one another with respect to the chemical composition and gel strength: 1) the classical lambda-carrageenan which is soluble at all KC1 concentrations and does not gel. 2) the iota-like intermediate fraction which precipitates with KC1 at concentrations between 0.125M to 2.0M and does not gel. 3) kappa-carrageenan which precipitates at a concentration of 0.125M KC1 and does form gels. 37 Table 3. Preparation and properties of carrageenan fractions from Chondrus crispus. Gel Strength Fract %3,6-AG %S0 Na * in ion No. Cone, of KC1 Yield (w/w) Iw/w) (n) mil)t(l) 1 Precipitate at 0.0625 M 48 29.2 24.8 9.4 230 2 Precipitate between 0.0625 and 0.25M 20 27.0 32.2 8.6 40 3 Soluble at 0.25M 31.4 9.0 39.6 9.2 0 3A Precipite from N o . 3 at 1.5 M 15.1 15.0 34.3 3B Soluble part of No.3 at 1.5 M 16.3 4.0 40.0 * Intrinsic viscosity (1) All yields given as per cent of total recovery (ca. 90%). From Pernas et al., (1967). 38 Depending on the desired functionality of the final extracts# the fractionation method can be far more detailed than those mentioned thus far* For example, Lin (1971) in his study on the hydrocolloid/protein interaction suggested a method of fractionation of potassium sensitive, potassium insensitive, potassium/calcium sensitive, and potassium sensitive/calcium insensitive fraction from the initial crude carrageenan extracts of Chondrus crlspus at 90C. He employed the classical KC1 to obtain the potassium sensitive fractions which received further treatment with CaC12 to prepare the potassium sensitive/calcium sensitive carrageenan fractions. A similar approach appears in the report of Kardosh (1975). In addition to the types of salts and their concentration in the extraction media, other parameters are also known to greatly affect the properties of the extracts. In the presence of an oxidizing agent such as hypochlorite, carrageenan extract, the kappa-fraction in particular, undergoes depolymerization to give products of low intrinsic viscosity and high sulfate content(Masson, 1954). Mild acid treatment of the carrageenan solution presents a similar degradation problem caused by the cleavage of the acid labile 3,6-anhydrogalactosidic linkages (Black et al., 1965). On the other hand, the lambda-carrageenan fractions which are lacking in the 3.6 anhydrogalactose group are 39 expected to be more resistant to mild acid hydrolysis. Unbuffered extraction media, where water is the only extraction solvent, display similar degradation behavior since the heat treatment of the extractant accompanies a decrease in pH which leads to the acid hydrolysis {Masson, 1954). According to the study of Masson et al., (1955), the maximum stability of the extracts during the fractionation process can be obtained at pH 9.0 in the presence of salts. They further suggested that the extraction be conducted in an inert atmosphere to avoid the incorporation of the dissolved air which accelerates the degradation of the polymer. A supporting view was later postulated by 0esai(1979) who found that the thermal degradation of carrageenan was significantly reduced when oxygen in the solution was removed by argon flushing. Alkaline extraction condition appears to be important , not only for the chemical stability of the extractant, but also for the conversion of the non-gelling lambda-fraction to the gel-forming theta-carrageenan (Stanley, 1970). The intermediate fraction which precipitates in 0.125M to 2.0M KC1 solution also significantly increases the gel strength upon the alkaline treatment (Matulewicz and Cerezo, 1980). b). Variations in the extraction temperature. Extraction of carrageenan is highly dependent upon 40 temperature. Fixing the ionic strength at 0.02 with aqueous sodium acetate as an extraction solvent, Goring and Young (1955) investigated the effect of temperature on the properties of carrageenan extract. They heat-treated dried and ground Chondrus crispus at 30, 60, and 120C successively. The extraction fractions differed widely in gel strength in decreasing order of the 60C extracts > 120C > 30C, and in intrinsic viscosity with the 60C fraction markedly higher than the other two. The common property that the workers observed was polydispersity of the molecular weight of each fraction. Later in 1975, Kardosh recovered carrageenan fractions from the dried seaweed, Chondrus crispus, in aqueous media by successively applying the extraction of the material at increasing temperatures from 3C to 90C. He reported that: More than 70% of the total extracts was obtained at temperatures between 30 and 50C. Zonal electrophoretic patterns of the fractions indicated that low temperature extracts (3-22C) possessed characteristics of lambda-fractions while the medium to high temperature (31-80C) fractions showed properties more similar to the kappa and iota varieties. Using a sodium phosphate buffered media at pH 7.0 and ionic strength of 0.1, Renoll and Hansen (1979, unpublished) further studied the sequential extraction method at varying 41 temperatures* The molecular size of the fractions was apparently temperature dependent; the intrinsic viscosity of the fractions above boiling temperatures was significantly lower than that of the medium temperature groups (60-80). Recently the optimum temperature and time conditions for extracting sulfated polysaccharides (SPS) from various species of Ulvacea were investigated by Yamamoto (1980B). He obtained SPS fractions at 20, 40, 60, 80, 90 and 1200 successivel. Low intrinsic viscosities were demonstrated for fractions obtained at both low and high temperatures. He interpreted the low viscosity as a result of the autolysis by the action of enzymes at the low temperature and by thermal decomposition at the high extraction temperature. The medium to high temperature fractions (80-90C) showed higher molecular weight and higher yield than the low temperature extracts (30-40C). Using the red algal seaweed, Eucheuma spinosum, with water as a solvent, the carrageenan recovery from the iota-fraction rich plant was studied by Tong et al.(1980, 1981) using 4C to extract fractions and using 100C to extract carrageenan from the remaining fractions. At 4C, a major fraction of high sulfate content was recovered while at 100C a minor component of low sulfate component was extracted* The average molecular weight of the 100C extracts was greater than that of 4C fractions. Upon comparing the average 42 molecular Bize of the 4C fraction with kappa-carrageenan, they learned that the 4C extracts of Eucheuma Bplnoaum consisted of molecules much smaller than those of kappa-carrageenan and proposed that the higher solubility of the Eucheuma extract in dilute potassium chloride solution, compared to kappa-carrageenan, might be due to the smaller molecular size. In the extraction of carrageenan, excess heat treatment at a high temperature can cause heat degradation of the extracted polymers. According to Masson (1954), the polymer size of the extract, as measured by the intrinsic viscosity, was drastically decreased with the time of heating at 90C. Similar findings were reported by Goring and Young (1955) and Black et al., (1965). Masson (1954) also reported that heat degradation adversely affects the molecular weight of the polymer and proportionally increases the reducing power of the carrageenan. He proposed from kinetic studies that heat degradation involves two stages: a) an initial rapid degradation stage where about 0.3% of the complete hydrolysis takes place b) a first-order random degradation which becomes important at temperatures above 60C. 43 B. The Stabilization of Calcium Sensitive Milk Proteins by Carrageenan. Casein is defined as "those phosphoproteins that are precipitated from raw skim milk by acidification to pH 4.6 at 20C" (Whitney et al., 1976). In milk, casein exists as colloidal particles (casein micelles) in which the subunits, mainly alpha-, beta-, and kappa-caseinB, are interconnected in the ratio of 3:2:1 respectively (Farrell, 1973) to form a spherical particle of about 100 angstrom(A) in diameter (Shimmin and Hill, 1964). These subunits differ in the number of phosphate residues, and show different electrophoretical mobility (Whitney et al., 1976) and stability against precipitation by calcium ion (Linderstrom - Lang, 1929). When treated with 10 mM calcium chloride in the neutral pH range, the calcium sensitive proteins, alpha(sl) and beta-caseins, tend to precipitate while the calcium insensitive kappa-casein remains soluble. Zittle (1961) demonstrated the ability of kappa-casein to stabilize the calcium-sensitive alpha(SI)-and beta-caseins. Thus, kappa-casein is the key to the stability of casein micelles (Farrell, 1973) against precipitation by calcium ion under the normal milk condition of pH, ionic strength, Ca++ ion concentration ( 2B to 30mM), and temperature. The formation of caBein micelles, however, does require the presence of 44 calcium ions (Farrell and Thompson# 1973) through which kappa- and alpha(Si)-caseins undergo ionic bonding leading to micelle formation. In forming the casein micelle# the exact mode of the physical arrangement of the subunits has not been fully elucidated although several models have been proposed (Shimmin and Hill# 1964; Morr et al.# 1971). These models are compared in the review of Farrell and Thompson (1974). For example, on the basis of disruption patterns of casein micelles, Morr et al.# (1971) proposed! The alpha(Sl)-# beta-, and kappa-monomers may be aggregated by calcium into small subunits which are stabilized by hydrogen bonding and calcium caseinate bridges, and these subunits# in turns, are aggregated into micellar structure by colloidal calcium phosphate. As for the localization of kappa-casein in the micelle, Horisberger and Van Lanthern (1980) from their ultrastructural study of lectin-labelled bovine casein micelles speculated that The glycosylated kappa-casein is mainly located in the bridging network interconnecting the micelles and appear to be loosely associated with the micelles. It has been shown that the stabilizing role of 45 kappa-c&Bein for the calcium-sensitive alpha(SI)- and beta- caseins can be duplicated by a selected group of sulfated polysaccharides, such as carrageenan and furcelleran (Hansen, 1968; Lin, 1977). Hansen (196B) demonstrated that diluted model systems of calcium-sensitive casein can be stabilized against calcium-induced precipitation by kappa-carrageenan and less effectively by lambda-carrageenan* Chakraborty and Hansen (1971) observed by electronmicroscopy that kappa-carrageenan formed stable casein micelles which resembled in many ways the ultrastructure of the alpha(SI)-casein/kappa-casein complex. The formation of the carrageenan-protein micelle also requires calcium ion as in the case with kappa-and alpha(SI)-casein complex (Lin and Hansen, 1970; Skura and Nakai, 1980). The interaction products of alpha(SI)-casein with kappa-carrageenan was observed to be more stable against increases in calcium concentration and heat treatment than the kappa-casein/alpha (SI)-casein complex at neutral pH. The diluted model system of skim milk and carrageenan also exhibited stability against coagulation by calcium ion following rennet treatment (O'Loughlin and Hansen, 1973). The effectiveness of the stabilization by carrageenan varies widely depending on the physical and chemical nature of the polysaccharides. Lin and Hansen (1970) with 46 supporting evidence by other investigators proposed a number of factors which may affect the effectiveness of the protein stabilizers; 1) molecular size (O'Loughlin and Hansen, 1973; Kardosh, 1975) or molecular weight (Snoeren, 1976). 2) conformation of structural sugar units (Chakraborty and Hansen, 1972). 3) chemical composition of the sugar units with respect to sulfation (Lin and Hansen, 1970; Lin, 1977). Chakraborty and Hansen (1971) speculated that a certain chain length of cjarrageenan in double helix zones (Fig. 10A) separating the casein-carrageenan complex was necessary for the carrageenan to be effective in the protein stabilization. Low molecular weight(<100,000) as well as high molecular weight (>300,000) failed to provide proper stabilization of the calcium sensitive protein in skim milk. Kardosh (1975), while evaluating the carrageenan fractions extracted from Chondrus crispus, noticed that moderate heat degradation of high molecular weight fraction (rich in kappa-carrageenan) considerably improved its ability for protein stabilization. However, strongly degraded carrageenan possesses no stabilizing activity (O'Loughlin and Hansen, 1973). Snoeren (1976) demonstrated that the stabilizing ability of kappa-carrageenan increased with the 47 • • « 4 • • * «»«»»» m w • .* . • • :;o u « C/\) ( f c ) Fig 10. The interaction of K-carrageenan with Lsl-casein in the presence of calcium. (A) Internal structure of carrageenan-alpha(si)-casein aggregates separated by ordered double helix zones. (B) General view of carrageenan/casein interaction. (Adapted from Chakraborty and Hansen, 1972) 48 molecular weight of the polysaccharide with a minimum molecular weight of 100,000 daltons to be the critical point for stabilization. The conformation of structural sugar units also greatly affect protein stabilization against calcium induced precipitation. Lin and Hansen (1970) speculated that the carrageenan varieties, such as kappa- and iota-carrageenans, with the conformation which can promote gel formation can stabilize the proteins from precipitation by calcium more effectively than non-gelling lambda-carrageenan. For example about twice the quantity of lambda-carrageenan was necessary to achieve the same degree of stabilization as exhibited by kappa-carrageenan (Hansen, 1968). However alkaline conversion of non-gelling lambda-carrageenan into gelling theta-carrageenan markedly improves its ability for protein stabilization (Chakraborty and Hansen, 1971; Matulewicz and Cerezo, 1980). In addition to the carrageenan varieties, other sulfated polysaccharides, such as fucellaran, and heparin, though not effectively, stabilize the calcium sensitive milk proteins. Upon Bulfation, the neutral polysaccharide, locust bean gum, can also be a powerful stabilizer similar to carrageenan (Lin and Hansen, 1970} Lin, 1977). In a processed milk system, the hydrocolloid-protein interaction has been an important functional property in 49 stabilizing not only the calcium sensitive proteins but also the emulsion system as a whole. The mechanism of the stabilizer-protein interaction in a processed milk system such as evaporated and chocolate milk has been well elucidated (Chakraborty and HanBen, 1971; Snoeren, 1976). Hansen (1982) speculated that two different types of mechanisms are involved in the hydrocolloid-protein interactions: 1) a general type of electrostatic association which is governed by the availability of ionizable groups of opposite charge. 2) formation of micellar structures between specific hydrocolloids and calcium-sensitive proteins. The first type of mechanism# electrostatic association between hydrocolloid and protein# includes both general (Hidalgo and Hansen, 1969) and specific association (Snoeren, 1976). Sulfated polysaccharides such as carrageenan and furcelleran, are negatively charged over a wide range of pH#* therefore# the electrostatic interaction between the hydrocolloid and protein becomes very important in low acid food where protein is net positively charged (pH < pi). A similar type of ionic interaction can account for the formation of insoluble complexes between beta-lactoglobulin and the weakly acidic 50 carboxymethylcellulose at pH 4 (Hidalgo and Hansen, 1969). Above the isoelectric point of protein (pH > pi), both the protein and the sulfated polysaccharide have net negative charges, therefore, the electrostatic interactions involve polyvalent metal ions such aB Ca2+ in solution which bridge the negatively charged carboxyl groups on the protein and the sulfate ester groups on the polysaccharide (Stanley, 1970). In the absence of the divalent metal ion, the complexing between the negatively charged polysaccharides and proteins can also take place via specific electrostatic association involving only a part of each polymer. Snoeren (1976) demonstrated that the specific electrostatic attraction between kappa-casein and kappa-carrageenan can occur when at neutral pH provided the net positively charged amino acid residues from 20 to 115 of the kappa-casein region are oriented toward the sulfate ester groups of kappa-carrageenan (Fig. 11). The principle of a localized re/ion of strong positive charges providing a site for interaction may be the basis of the chromatographic purification of proteins using kappa-carrageenan in affinity chromatography (Snoeren, 1976, Chibata et al., 1981). In preventing the phase separation of evaporated milk, Snoeren (1976) suggested that these types of specific electrostatic interactions between kappa-carrageenan and kappa-casein is required at the initial stage followed by the sol-gel Fig 11. The sol-gel transition as proposed for kappa-and iota-carrageenan in milk. From Snoeren, 1976 52 transition(Fig. 11) o£ unassociated kappa-carrageenan to form a three dimensional network* In much the same way. chocolate milk can be stabilized against the phase separation (sedimentation of cocoa particles) by the formation of the weak kappa-carrageenan gel network in which the suspended cocoa particles are entrapped (Snoeren, 1976) However, according to Hansen ( 1 9 8 2 ) , this may not be the major mode of action for the stabilizer in sterilized concentrated milk products where kappa-casein has undergone heat-induced interactions with beta-lactoglobulin and where the casein micelles have been disrupted by fat-protein complexation. In those emulsion systems, the second type of hydrocolloid-protein interaction mechanism which involves the formation of micell structures between the polymers may well account for the emulsion stabilization. It is known that carrageenan can form a stable micelle with alpha( s i ) -casein (Hansen, 1 9 6 8 ). Chakaraborty and Hansen (1 9 7 1 ) in their study with a model system of alpha(Sl)- casein solution proposed that kappa-carrageenan -alpha(Sl)-casein interaction features entrapment of small, calcium aggregated bodies (complex) in the non-helical region of carrageenan (Fig. 10A) and the non-associated part of carrageenan provides effective physical separation of individual particles (Fig. 10B) that would have otherwise interacted to form large, colloidally unstable precipitates. 53 They suggested that the protein reactivity of carrageenan would be limited to the uncoiled helix-free 'zones where intense ion-protein aggregation takes place while the ordered double helix zones separate the kappa-carrageenan/alpha(SI)-casein aggregates which may be viewed as stable carrageenan/alpha(SI)-casein micelle (Hansen, 1968). Later, Skura(1976) proposed that similar type of physical entrapment of calcium-alpha(SI)-caseinate particles within the kappa-carrageenan network leads to the stabilization of alpha(Sl)-casein. Therefore, the stabilization of evaporated milk against creaming may, as Hansen (1982) suggested, involve the interaction of carrageenan with casein micelles on the fat globule surface leading to the formation of carrageenan/calcium sensitive protein aggregates which are then entrapped in the carrageenan network. 54 C. Molecular Weight and Size of Carrageenan* « Carrageenan is a polydisperse polymer with respect to molecular weight and size. A number of molecular weight and size properties reported by different investigators are listed in Table 4. It is apparent that uncertainties exist in the assignment of correct molecular weights for these polymers and different results have been obtained with different methods. Gel permeation chromatography has found application for determining the molecular weight distribution of the polymer (Bathgate, 1970; Vollmert, 1973; Yamomoto, 1980B; Szewczyk, 1981). It is based on the diffusion of the polymer molecules into the capillary interstices of a cross-linked, swollen polymer gel, which permits a sharp separation into fractionations of different molecular weights (Vollmert, 1973). Using porous silica gel, Marine Colloids (1972) reported the weight average molecular weight of calcium carrageenan (Seakem 2) to be 370,000 daltons, which is a calculated theoretical value obtained from the molecular weight distribution curve. Tong et al., (1980, 1981) employed a Sepharose-4B gel column with a molecular exclusion limit of 5 million daltons to examine the 4C and 100C extracts of Eucheuma spinosum. The 4C extracts showed weight average molecular weights between 1.5 and 2.0 55 Table 4. Molecular Weight and Size Properties of Carrageenan Intrinsic viscosity Carrageenan Ionic Molecular Weight Sample______dl/g Strenq. Mw Method Reference Kappa, Sedim- 0.125M KC1 Precipitate ention & Vise- Smith et 7.5 0.50 290.000 osity a l .(1952) Lambda , 0.125M KC1 Soluble 11.1 0.50 690.000 Kappa (K-salt) Water only 42.0 0.0 - 20% glucose Elfak et solution 25.0 a l .(1978) Light Vreeman Kappa Scatt et a l ., Ho. 6.83 0.10 604.000 ering (1980) HMR 7.15 0.10 522.000 Kappa(Na) 12.0 0.01 430.000 Osmo Rinaudo et metry al.(1979) Iota(Na) 15.2 0.01 390.000 Lambda(Na) 17.0 0.01 710,000 Gel Fil tration Ultrace- Marine Kappa, ntrifug- Colloids Seakem 2 9.9 0.10 370,000 ation (1977) 4C Extract of Eucheuma 1,500,000 Gelfil- Tong et spinoBum 76.92 ? tration al.(1980) alSolution of NaCl. 56 million, while the 100C extracts showed an even higher value with large proportions toeing eluted at the exclusion limit of 5 million. In comparison with values previously reported (Cook et al., 1952; Rinaudo et al., 1979; Vreeman et al., 1980), these results are exceptionally high. For example, Rinando et al., (1979) using osmometry obtained molecular weight values of 430,000 daltons for kappa, 390,000 for iota, and 710,000 for lambda. Other techniques for the determination of molecular weight and size properties of carrageenan include viscometry (Smith et al., 1943; Elfak et al., 1978; Yamamoto, 1980B), refractometry (Batsanov, 1961; Vreeman et al., 1980), osmometry (Rinaudo et al., 1979), light scattering (Rinaudo et al., 1978; Vreeman et al., 1980), and analytical ultracentrifugation (Cook et al., 1952; Skura, 1976; Desai, 1979). The viscometric method with special reference to intrinsic viscosity is often used to determine the molecular size of macromolecular compounds. It is based on the principle that the relative viscosity of a polymer solution (or dispersion) of spherical, colloidal particles depends on the hydrodynamic volume occupied by the dissolved particles (Vollmert, 1973). The larger the occupied volume, the higher the intrinsic viscosity and the larger the molecular size (or molecular weight) of the polymer. It is known that the intrinsic viscosity of non-globular polymers is 57 influenced by a variety of factors which include solvent (Ullman, 1981), temperature, structure of the polymer chain (Van Holde, 1967), and ionic strength (Smidsrod et al., 1980). Table 4 lists the intrinsic viscosity values of carrageenan varieties at different ionic strength of sodium chloride. At zero ionic strength, kappa-carrageen showed the highest viscosity value, 42 dl/g (Elfak et al., 1978), with much lower numbers reported for carrageenan dissolved in electrolyte solutions, e.g., 12.0 dl/g, for 0.01 ionic strength (Rinaudo et al., 1979). A high value for intrinsic viscosity at low ionic stength may be expected on the basis that the many negatively charged groups along the polymer chain repulse each other electrostatically in the absence of a dissolved electrolyte, resulting in an expansion or stretching of the coil, leading to an increase in the hydrodynamic volume occupied by the solvated polymer (Vollmert, 1973). As the ionic strength of the solvent increases, the polymer coil generally contracts following the shielding of the charged groups in the chain. Smidsrod et al., (1980) reported , however, that at a sufficiently high level of ionic strength (1 > 0.50), the intrinsic viscosity of iota-carrageenan increased markedly with the ionic strength when gel promoting cations were used including potassium chloride, sodium chloride, and lithium chloride. They 58 suggested that this phenomenon must be due to either a conformational change in the isolated molecules (intramolecular transition) or a very limited, intermolecular association, such as dimerization. It may be expected that intrinsic viscosity values of carrageenan would increase with the degree of sulfation of the polymer because of the increase in intramolecular repulsions* The values by Rinaudo(1979) (Table 4) show an increase in the order of kappa< iota< lambda. However, these differences may alBo be the result of differences in molecular weight. The effect of glucose addition on the intrinsic viscosity of solutions of kappa-carrageenan was studied by Elfak et al., (1978) who reported lower values in the precursor of the neutral sugar and suggested that the dissolved sugar lowered the dielectric constant as well as the availability of water for hydration of the polymer, causing a less extended configuration of the carrageenan. The molecular weights of polymers can be related to their refractive index as suggested by Batsanov (1961) from the equation proposed initially by borenz-Lorenz* n**2 - 1 M 4 ■ ■ ■ ■ — — ■ Rm » ■ %, No Odm n**2 + 2 D 3 59 where "M" ie the molecular weight, "D" the density of the medium, "No" the Avegadro number, "OCm" the molar polarizability, and "Rm" the molar refractivity which is affected by the chemical composition and molecular arrangement of the substance being examined (West, 1954). By using X-ray diffraction methods, molar polarizabilities of a macromolecule can be obtained which then allows the computation of a refractive index, as shown by Orttung and Armour (1967) with amino acid crystals. The refractive index of crystalline substances can usually be determined by polarizing microscopy using the techniques of dispersion staining (McCrone et al., 1978) or the Becke line method (West, 1954). Using the modified Lorenz-Lorenz equation, Rhein and Dawson (1971) correlated the inverse number average molecular weight with the refractive index for the polymers of isobutylene and ethylene oxide. Although other natural fibers of plant origin such as cotton and silk have been characterized by their refractive indices (Kirchgessner and Grisser, 1965), carrageenan in the solid state has not been a subject of refractometric analysis. However, the refractive index of the aqueous carrageenan system has often been measured in terms of a refractive index increment (Vreeman et al., 1980). Carrageenan extracts at 60-120C of 60 Chondrus crispus showed an increment o£ 0.132 ml/gr (Gordon and Young, 1955) while Vreeman et al. (1980) reported 0.118 + 0.003 ml/gr for commercial kappa-carrageenan samples. The BUlfated polysaccharide extracts of Ulva species showed 0.099 and 0.100 ml/gr for Ulva pertusa and Ulva conglobata respectively (Yamamoto et al., 1980B). As a method to account for differences in the degree of sulfation and molecular size of carrageenan, electrophoresis is a simple and reliable method. The electrophoretic method is especially useful for separating, identifying, and measuring individual polysaccharides present in the mixture of polysaccharides (Fuller and Horthcote; 1956, Hidalgo and Hansen, 1968; Breenan et al., 1976). Effective electrophoresis of the polymers with reference to the qualitative separation of a mixture, however, requires proper buffer conditions of viscosity, ionic strength, and pH (Hidalgo and Hansen, 1968). Cook et al. (1952) reported that the electrophoretic mobility of hot water extracts of Chondrus crispus was 12.8E-15 cm**2/volt.sec at X«0.10 - 0.15. Kappa- and lambda-carrageenan fractionated by 0.125M KC1 precipitation showed electrophoretic mobilities of 14.6 and 15.8E-15 cm**2/volt.sec respectively (Smith et al., 1954). The electrophoretic separation of a mixture of anionic stabilizers, kappa- and lambda-carrageenan, carboxymethyleellulose, and alginate was studied by Chang et 61 al., (1974) who found that in borate buffer at pH 7.0 the food Btabilizers exhibited nearly the same mobilities, but a complete separation was achieved in malonate buffer at pH 2.9. The ralative mobility of kappa-carrageenan, they determined, was 0.70 in relation to lambda carrageenan. Hansen and Renoll (1974) observed that the zonal electrophoresis patterns of carrageenan stabilizers of heat processed milk products were significantly altered from the patterns of their respective controls. The electrophoretic pattern, due to the heat treatment, was almost identical with that of carrageenan degraded by controlled acid hydrolysis. 62 D. Molecular Weight Determination by Analytical Ultracentrifugation. The analytical ultracentrifuge may be used to generate physical constants for polymers such as sedimentation coefficients, S^p ,w, and diffusion constants, d£q *w , on the basis of which the molecular weight of the polymer can be calculated. Table 5 lists values obtained for carrageenan. Sedimentation velocity of lamda-carrageenan appears to be faster than that of kappa-carrageenan (Smith et al., 1954). The sedimentation coefficients of carrageenan showed an increase with the temperature as reported by Goring and Young (1954). In ultracentrifugal determination of the molecular weight of biological polymers such as carrageenan, the technique of either sedimentation equilibrium (Badui et al., 1978; Desai, 1979; Yamamoto, 1980A) or sedimentation velocity coupled with diffusion analysis (Cook et al., 1952) have been employed. Svedberg (1940) stated that the equilibrium method, even though it involves laborious calculations, has the advantage of requiring only the determination of the concentration distribution in the rotating column of solution at equilibrium. The average molecular weight of carrageenan varieties determined by the sedimentation equilibrium method are shown 63 Table 5. Centrifugal Analysis of Carrageenan. Sedimen "Di-ffu-- ■ ■ tation sion Coeffic Const Carrageenan ient ant Sample S(20C) D(20C) Mw Reference x(10E-13) x(10E-7) Hot water Cook et al. Extracts 3.67A0.17 1.41 120,000 (1952) kappa, 0.125MKcl Smith et precipitate 4.6 0.82 290,000 al (1954) Lambda, 0.125M Smith et KC1 Soluble 7.7 1.22 690,000 al., (1954) Stanley and Iota 230,000 Renol(1974) Kappa, 0.4&KC1 13 Goring and precipitate (5 40, W) 1 ,000,000 Young(1955) Lambda, o.4%Kcl 16 Goring and) Soluble (S 49, W) 1,400,000 Young(1955) Kap p a , 0.2M KC1 4.40 - Lin (1971) Kappa, acid Elfak et Hydrolysed 4.82 - al., (1978) Seakem (Type 2) Marine 213,000 Desai(1979) Kappa 296,000 Lambda 761,000 64 in Table 5. Some descrepancies are again evident between the values from different studies. It has been shown that the apparent weight average molecular weight, Mw(app), of carrageenan by the equilibrium method can differ widely depending on the concentration and rotor speed (pressure) similar to the effect noticed earlier by Wales et al.(1946) for bovine serum albumin. Badui et al.(1978) found that Mw(app) of carrageenan (Seakem 2) decreased linearly with the rotor speed. For that reason, they suggested that a reversible, pressure-dependent dissociation of the carrageenan polymer takes place such as observed by Josephs and Harrington (1967) in their ultracentrifuge study with myosin. Skura (1976) later observed concentration-dependence of sedimentation rate with kappa-carrageenan. In order to compensate for the concentration- and pressure-dependence of the sedimentation behavior of carrageenan, Desai (1979), in his equilibrium study with kappa- and lambda-carrageenan, obtained corrected molecular weights, Mw(corr), by extrapolation to zero speed and zero concentration. His values of 296,000 daltons for kappa-carrageenan and 761,000 for lambda-carrageenan agree with the particular values reported by Smith et al.(1952) and Rinaudo et al.(1979). Presure effect (Howlett et al., 1970) and concentration effects (Williams, 1972) occurring during sedimentation 65 equilibrium can be advantageously used to investigate the assocation-disBociation behavior of protein molecules. These effects however, make the interpretation of the ultracentrifugal data more difficult, particularly with respect to calculation of a true molecular weight distribution. In their study with myosin, Harrington and KegeleB (1973) showed that pressures of the order of 100-500 atm are generated at the base of a centrifuge cell at high rotor speeds which leads to possible changes in the aggregation state of various polymers. For example, under moderate pressure, macromolecules, such as myosin and sea urchin ribosomes, undergo depolymerization (Josephs and Harrington, 1967). For pressure and concentration dependent polymers it is still possible to derive the corrected molecular weight corresponding to a non-stressed condition and zero concentration. Fujita (1975) derived a mathematical equation for the determination of the molecular weights of multiple component polymers in non-reacting systems in which terms were included for concentration and pressure effects. In the velocity centrifugation of random coil polymers, the linear and second-order pressure effects have been investigated by Mulderije (1982) who used polystyrene in a cyclohexane solvent. In the power series expansion of l/S with respect to pressure, the linear pressure effect was 66 found to increase linearly with the product of concentration and molecular weight. He interpreted the concentration dependence of the pressure effect as an effect of the pressure on the thermodynamic interaction. MATERIALS AND METHODS The current study has dealt with the physical, chemical, and functional characterization of carrageenan fractions obtained from dried seaweed,Chondrus crispus. The experimental plan is outlined in Fig. 12. A. Carrageenan Samples 1. Calcium carrageenan (Seakem #2), lot <152306, dated 5/7/76; Manufacturer-Marine Colloids, Inc., Springfield, New Jersey. 2. Carrageenan fractions from dried Chondrus crl3pus were obtained by the following method. B. Sequential Extraction of Carrageenan from Chondrus crispus 1. Buffer preparation: pH 7.0, ionic strength <>0.1 sodium chloride 5.84 gram/2 liters NaH2P04 H20 2.12 Na2HP04 4.04 2. 10.0 grams of dried seaweed, Chondrus crispus, were first extracted in the buffer overnight at 6C. Then a 2 hour-extration at each step was done successively in 10C intervals from 20 to HOC. G7 68 Dried Seaweed Chondrus crispus* I Sequential Extraction at Successively Elevated Temp(5-110C). Eleven Carrageenan Fractions 1 1 Physical Characterizationi Chemical Characterizations Gel Filtration KCL Precipitation Analyt. Ultracentrifugation Zonal Electrophoresis Intrinsic Viscosity Refractive Index Functional Characterizationj A lpha (SI)-Casein Stabilization Fig. 12 Experimental Outline o£ Physical Chemical and Fuhctional Characterization of Carrageenan Fractions of Chondrus crispus 69 Fraction Temp Time Buffer tfumher ( 1 5 overnight 500 2 20 2 300 3 30 II tl 4 40 41 II 5 50 IIII 6 60 IIII 7 70 II t l 8 80 II II 9 90 II II 10 100 II 275 11 245 F II 200 3. After the extraction, the extrants were centrifugedi Fractions No. 1-4 in Lourdes centrifuge No, 1 and 2 i cold rotor for 10 min. No. 3 and 4 : room temperature rotor for 30 min. Fractions no. 5-11 in Sorvall centrifuge No. 5 and 6: room temperature No. 7 - 11 : refrigeration unit on 4. Supernatants from the centrifugation were decanted and dialyzed against deionized water in a cold room (44-46 F) for at least 24 hours with two water changes, the first change after 4 hours and the second change overnight. 5. The Conductivity of the sample solutions was checked on an LKB conductolyzer, with a dip cell having a cell constant of approximately 5.00. Dialysis was continued until the conductivity was less than the limit of the 70 instrument (< 5*10E-4 mhos). 6 . The dialyzed samples were then centrifuged to remove impurities (10,000rpm, 60min, 0-10C). 7. The supernatant from the centrifuge was decanted and freeze dried and kept in cold storage until used. C. del Filtration Gel filtration was carried out on a 2.6 x 67cm column of Sepharose 4B (Cross-Linked) (Pharmacia Fine Chemicals) with an effective fraction range (for globular protein) from 8*10E4 to 10E6 daltons. The column was pre-equilibriated with 0.5M NaCl buffer before each run. The column was eluted at a flow rate of lOml/hr. The column temperature was maintained at 60C in order to ease the flow of the viscous fractions. The column was calibrated with respect to the elution volume by using the molecular weight standards of Dextran T10 (10,000 daltons), T70{70,000 daltons), 7500(500,000 daltons) and Blue Dextran T2000(2,000,000 daltons). Extraction samples of 20mg were applied in a volume of 5ml (0.4% solution). Fractions of 5ml were collected? 0.5ml samples were analyzed for carbohydrate content by the Phenol-Sulfuric Acid method. The weight average and number average molecular weight of each fraction were obtained as described by Vollmert (1973). 71 D. Ultracentrifugal Analysis Ultracentrifugation of each fraction was carried out at 35C in a Spinco Model E Analytical Ultracentrifuge equipped with RTIC-unit, electronic speed control, and Schlieren- and interference-optics. in this experiment, the sample preparation and the interpretation of the ultracentrifugal pattern followed the methods of Desai(1979). From the apparent weight average molecular weights, a corrected molecular weight, Mw(corr), of a fraction was obtained by extrapolating to zero speed and concentration by multiple regressison analysis. E. Viscosity Measurement A solvent buffer of 0.25M sodium chloride and 0.05M cacodylate at pH 7.0(adjusted with 50%(w/w) HaOH) was used to prepare a 0 .3 % solution (w/w) of the carrageenan fractions. For the viscosity measurement, a Cannon-Ubbeholde Dilution Viscometer (Ho. 150, E 583) was used. After the first three readings of the efflux time of the 0.3% carrageenan solution, dilution of the solution was made in the viscometer bulb successively from 0.170, to 0.120, 0.092 and 0*075% as illustrated in the following table. 72 Sample Buffer Final Volume Concentration ml ml ml % a 0 8 0.300 a 6 14 0.170 14 6 20 0.120 20 6 26 0.092 26 6 32 0.075 The mixing of the sample solution following each addition of solvent was accomplished running a stream of filtered air into the bulb. The measurements were carried out at 38C +0.2 and the average value of three different readings was used to calculate the viscosity for a solution. Prior to charging with the new sample solution, the viscometer was washed sequentially with hot water, distilled deionized water, 30% (w/v) sulfuric acid, distilled deionized water, acetone, and finally dried with an air stream. F. Refractive Index Determination. The refractive indices of the carrageenan sample fibers which were freeze-dried were estimatedd by the Becke Line method (West, 1954) in the following manner. 1. The bright field condenBor was installed and the light microscope was adjusted in focus. 2. A few small sample fibers were placed on a clean 73 glass slide and covered with a cover glass. 3. A suitable mounting medium (Cargille refractive index liquids) of a known refractive index was selected, and a drop of the medium was introduced at the edge of the cover glass, then allowed to flow under by capillary action. 4. The field of the particles to be examined was brought into focus. 5. The fine adjustment of the microscope was then used to raise the body tube. As the tube was slowly raised, a halo of bright light could be seen moving into or away from the crystal. If the halo moved into the crystal, the sample crystal had a higher refractive index than the mounting medium. If the halo moved into the medium, the sample crystal had the lower index of refraction* In other words, on focusing up, the halo (Becke line) moved into the medium of the higher index of refraction. 6 . Through repeated trials, a mounting medium was found which had nearly the same refractive index as the sample crystal. When a final match was approached, the particles would become nearly invisible, and in a perfect match would actually disappear completely from sight. When focused up, a yellow halo would be moving into the crystal while a blue halo would be moving out of the crystal. 74 Q. Zonal Electrophoresis The method developed by Hansen and Renoll (1974) was followed. A carrageenan solution of 0.4% (w/w) was applied to a cellulose acetate strip which was placed in the Millipore electrophoresis cell containing the buffer solution of calcium mlonate ethanol at pH 3.0. Electrophoresis was carried out at 100 volts for 15 minutes at room temperature. The strips were stained in a 0.2% Toluidine Blue solution containing 1% EDTA for two minutes and then destained with a 0.025M potassium acid phthalate wash solution. H. KC1 Precipitation of Carrageenan Fractions. The experimental design for the KC1 precipitation test is illustrated in the following table: Stock Solution: A : 0.4% carrageenan sample solution B : 0.25M KC1 solution for tube no. 1« 2, 3. 2.00M KC1 solution for tube no. 4, 5. Tube Ho. A H20 B ml ml ml Concentration 1 2 5 1 .03125M KC1 75 2 2 4 2 .06250 3 2 2 4 .125 4 2 5 1 .250 5 2 - 6 1.500 6 2 6 - 0.1% Carrageenan 7 1 7 - 0.05 Procedure t 1. Each sample solution was added in sequence into a 20 ml test tube and agitated for 1 min using a Vortex mixer* 2. The reaction medium was incubated for 30 min at room temperature to complete the reaction. 3. The reactant was weighed into a centrifuge tube of known weight after shaking 5-10 times on Vortex at the end of the reaction time. 4. The reactant was centrifuged at 17,000 rpm(26,000xg equivalent) for 5 min using rotor type #30 of Model L Beckman Ultracentrifuge. 5. After the centrifugation the supernatant was decanted and weighed in order to calculate the weights of the supernatant and the precipitate by difference. 6 . Sample volume of 0.2 ml supernatant was taken to determine carbohydrate content by the Phenol-Sulfuric method. In order to establish a standard absorbance curve, control carrageenan solutions of 0.05% and 0.1% were used. 76 Calculation: Precipitation - 100 * —- 1(■£&- . x^ j 3 W x — i - x i o o ) \ Slope Sample 1C J Abs ■ Absorbance reading o£ sample solution at 490mm. Slope ■ Slope of carrageenan solution standard curve (Unit : M o b IJIq , carrageenan). SW - Supernatant weight. Sample: Supernatant sample weight (^.2gram) used for the carbohydrate analysis. TC : Total weight of carrageenan in the reactant (8 ,000^ ) . 1. Stabilization of Calcium Sensitive Proteins. The ability of carrageenan fractions to stabilize calcium sensitive proteins was evaluated with alpha(SI) casein. The experimental method used by Lin(1971) was adopted after minor modification. The following table suggests the experimental design. Stock solution: 77 At 0.5% alpha(sl) solution at pH 7.30 Bx 0.05% carrageenan solution at pH 7.0-7.40 Ct 0.1M CaC12 solution Tube Ho. A B H20 C CHOtCasein ml ml ml ml Ratio 1 3 1 5 1 1 t 30 2 3 3 3 1 1 x 10 3 3 5 1 1 1 x 6 4 3 - 6 1 Calcium insensitive 5 3 - 7 - Total Protein 6 - 1 9 - 0.005% Carrageenan 7 3 7 - 0.015% 8 6 4 - 0.030% " 9 - 1 CaC12 control The solutions of carrageenan, alpha(Sl)-casein and water were added into a 2 0 ml test tube in sequence and mixed with the aid of the Vortex mixer. Upon the addition of calcium chloride solution, the mixture in the tube was agitated again using the Vortex for 1 min, then placed in the 30C water bath for 15 min. followed by centrifugation at 5500 rpm (about 3000xg) for 5 min. Supernatant of 3ml was mixed with 2ml O.IN-NaOH then UV absorbance at 280nm was read for the protein concentration. The carresponding absorbance readings of solutions of carrageenan control and calcium chloride were subtracted from values of the sample solutions. 78 Calculation! Wt absorbance reading of total protein X: 11 of calcium chloride control Y: 11 of carrageenan solution control Z " " of supernatant sample solution Z - ( X+Y ) %stabilization ■ ______x 1 0 0 W RESULTS A. Sequential Extraction of Carrageenan from Chondrus crispus. The functionality of carrageenan as a milk protein stabilizer is largely dependent on the molecular size and the chemical nature of its structural sugar units. The physical and chemical properties of carrageenan are in turn affected by the extraction method employed. The commercial production of carrageenan stabilizer is commonly carried out by cooking the seaweed at 90C under slightly alkaline condition. The resulting commercial extract contains all of the carrageenan material which can be solubilized under the extraction conditions. This research has dealt primarily with the physical and chemical charaterization of the extracts obtained from the sequential extraction of carrageenan from Chondrus crispus at gradually increasing temperatures from 5 to H O C and the underlying purpose has been to formulate extraction conditions which may yield highly functional carrageenan for use in the stabilization r* stabilized, fluid milk products. 1. Extraction YleldB of Carrageenan 79 80 The yields of carrageenan extracts sequentially obtained at gradually increasing temperatures are sh'own in Table 6 . The total weight of the carrageenan extracts obtained from 10 grams of Chondrus crispus was 5.46 grams (54.6% yield) which was somewhat lower than the yields achieved in previous studies (Renoll and Hansen, 1979, unpublished) under similar extraction conditions. However, the relative quantities were quite similar for the two experiments. The yields of carrageenan extracts at low temperature (5 and 20C) were small compared to yields at higher temperatures and the extract contained brown and red pigments. Major fractions with a combined yields of more than 40% of the total yield were obtained at 40C and 50C which is the range for the sol-gel transition of kappa and iota carrageenan. The remaining extraction fractions above 50C showed yieldB in the range of 7 to 9% with the exception of the 100C extract, part of which was lost by an experimental error. 2. Fractionation of Kappa-, lota-, and Larobda-Carrageenan The carrageenan extracts obtained from Chondrus crispus are heterogeneous chemical compounds composed largely of kappa-, and lambda-, and possibly iota-carrageenan in different molar ratios. Depending on the degree of Table 6. Yield of the Sequential Extract. 1 2 Extraction Yield Yield Temp., C Weight, % Weight, % 5 1 . 2 0 2.45 2 0 1.40 1.05 30 4.50 5.58 40 14.00 13.93 50 1 0 . 0 0 9.40 60 4.00 7.23 70 4.00 5.78 80 4.00 5.26 90; 5.00 6.42 1 0 0 2 . 0 0 6.06 1 1 0 4.50 4.00 Total 54.60 67.16% 1. Current recovery values. 2. Recovery values by Hansen and Renoll(1979f unpublished). 82 sulfation and the amount of 3,6-anhydro- -D-galactose-2- sulfate (3# 6 AO)in their repeating sugar units, the different types of carrageenan Bhow different solubility in potassium chloride solution which has been the baBis for the fractionation scheme advocated by Cook and Smith (1953). In order to determine the degree of chemical heterogeneity, each extraction sample was treated with potassium chloride. The precipitation percentages of each extract at varying concentrations of potassium chloride from 0.03I5M to 1.500M is tabulated in Table 7. Each carrageenan extract obtained at different temperature showed different solubility profiles (Fig 13) which was indicative of chemical heterogeneity. The precipitation behavior of the carrageenan extracts differed significantly for the extraction temperature as well as for the concentration of potassium chloride used as shown by analysis of variance (Table 8 ). Duncan's multiple range test was conducted in order to determine which pairs of means were significantly different (Table 9). The test indicated that each level of KC1 concentration yielded significantly different quantities of precipitate. When Duncan's test was conducted on the different carrageenan samples, the extracts obtained at the nine different temperatures could be grouped into five classes; Table 7. Precipitation(%) o£ the carrageenan extration fractions at varying potassium chloride concentrations EXt MOLARITY OF KCl X lo6 Temp.* C 3.15 6.25 12.5 25 150 3o 49.3 63.66 1S.XB 82.4 85.83 40 52.97 65.41 85.12 87.4 95.19 50 42.24 52.66 69.8 78.24 84.73 60 31.27 45.61 48.3 64.47 71.14 70 23.32 24.74 33.89 48.71 76.9 0 0 12.43 24.64 37.45 57.33 69.22 90 20.49 24.53 36.34 47.63 50.08 1 0 0 19.68 20.05 21.18 28.91 32.8 1 1 0 10.58 12.36 19.29 21.31 22.25 Seafcem 52.47 54.76 64.23 65.15 69.66 84 Table 8 . Analysis of Variancet Precipitation of the Carrageenan Extracts by Potassium Chloride. Sources S of MS P F.05 F.01 Total 26932 44 - - -- Extracts 17520 8 2190 39.05** 2.25 3.12 KC1 7617 4 1804 33.96** 2.67 3.97 Error 1794 32 56 - - - ** Significant at both p<.05 and p<.01 levels. 85 Table 9. DUNCAN1S MULTIPLE RANGE TEST: Precipitation of carrageenan extracts by Potassium Chloride. A. Molarity of Potassium Chloride. Means with the same letter are not significantly different. Alpha Level* .05 DF-32 MSi-56. 0901 Grouping Mean N Molarity oi Pot assium Chloride A 64.524333 5 '5 D 57.377778 9 4 C 47.394444 9 3 D 37.073333 9 2 E 29.142222 9 1 Carrageenan Extract Samples. Grouping Mean N Extraction Fractions A 77.218000 5 40 A 71.274000 5 30 A 65.534000 5 50 52.158000 5 60 D 41.512000 5 70 D 40.214000 5 80 D 35.814000 5 90 E 23.758000 5 1 0 0 E 17.158000 5 1 1 0 Table 10. Fractional corapbstion of Kappa, Iota, and Lambda-carrageenan in the Extraction Samples Obtained from Chondru 9 crispus. 1 Extracxtion Kappa(I) Iota Kappa(Il) Lambda 3,6AG Temp., C % % % % % 30 75.18 7.12 82.40 i7.6o 15.22 40 85.12 2.18 87.40 12.60 17.67 50 69.80 8.44 78.24 21.76 13.70 60* 48.30 16.17 64.47 35.53 9.79 70 33.89 14.82 48.72 51.29 7.29 80 37.45 19.88 57.33 42.67 10.19 90 36.34 11.09 47.63 52.37 7.45 1 0 0 21.18 7.73 28.91 71.09 3.35 1 1 0 19.29 2 . 0 2 21.31 78.69 1.70 Seakem 2 64.23 0.92 65.15 34.85 — 1. Concentration of 3.6 AG determined by Renoll and Hansen(1977, unpublished). Kappa(I) x 0.125M KC1 precipitate. Iota : 0.125M-0.25M KC1 precipitate. Kappa(II): Kappa(I) + Iota. Lambda : 0.25M KC1 soluble. PRECIFITRTION ru to D CO o FIG.13. C PRECIPITA CURVE N O I AT T I P I C E R P KCL GRIT F KCL OF MGLRRITT vs X V z X X£ * o + X A MOLARITY X1 IOC EX X100C EX EX EX SERKEM EX 50C EX EX FMO EX EX 80C 90C 60C 70C 30C 87 88 the 30C and 40C extracts contained more KCl-precipitable fractions than any other extracts while the 100 and HOC extracts contained primarily soluble forms. The precipitation results were then reevaluated for the fractional compositions of kappa-, iota-, and lambda-carrageenan in each extract by using the classification criteria:kappa as 0.125M KC1 precipitate, iota as 0.125M-0.250M KC1 precipitate, and lambda as 0.25M KC1 soluble. The contents of the three types of carrageenans present in each extract are listed in Table 10. As demonstrated by the extraction profiles in Fig 14, kappa-rich extractives were readily obtained in the range of 30-50C while lambda-rich fractions were released preferably er in the range of 100-110C. A transition in the pattern took place in the medium heat temperature range of 60-90C. The content of iota-carrageenan in the extracts did not vary greatly between the extracts even though the medium heat range extracts revealed the highest concentrations of iota carrageenan. The total weight (%) of kappa -f iota and lambda carrageenan in relaton to the total recovery of extracts were 66.36% and 33.4% respectively(Table 11), which are in good agreement with the previously reported values by Pernas et al. (1967) under similar experimental conditions. 89 A KflPPfl 4 IOTfl X LAMBDA VS TEMP D r\j a* 1 0 in 10 m co cn 20 29 30 47 56 65 75 84 93 102 111 120 EXTRACTION TEMP, C FIG. lU. FRACTIONAL COMPOSITION 90 Table 11. Fractional Yield of Kappa and Lambda as Per Cent of the Total Weight of the Carrageenan Extracts. Extraction Kappa-Carrageenan Lambda- Carrageenan Temp.* C % Total % Cum « Total 5 Cum Yield Total Kappa Yield Total Lambda (1 ) (2 ) (3) (4) 30 7.13 10.75 1.52 4.53 40 23.53 46.20 3.39 14.61 50 15.04 68.87 4.18 27.05 60 4.96 76.34 2.73 35.17 70 3.75 81.98 3.94 46.89 80 4.41 88.62 3.28 56.64 90 4.5B 95.52 5.03 71.61 1 0 0 1 . 1 1 97.19 2.73 79.74 1 1 0 1.84 1 0 0 . 0 0 6.80 1 0 0 . 0 0 Total 66.36 33.645““ 1. Kappa yield as % of the total weight of the carrageenan extracts. 2. Cumulative yield of kappa as % of the total extractable kap p a . 3. Lambda yield as % of the total weight of the carrageenan extracts* 4. Cumulative yield of lambda as % of total extractable lambda. 91 3. Zonal Electrophoresis. Electrophoretic separation of colloids on cellulose acetate is primarly a function of charge difference (Chang et al., 1974). Zonal electrophoresis of the samples was conducted to obtain additional information about the composition of the extracts. The results, shown in Fig 15, demonstrate that sequential extraction leads to distinctively different carrageenan materials. Samples extracted at 5C and 20C produced only weak patterns indicating an absence of strongly sulfated polysaccharides. The extracts obtained at 30C through 80C showed strong stationary bands and less distinctive moving bands which are characteristic features for the potassium and calcium sensitive carrageenans according to Lin(1971) and Chang et al. (1974). The extraction samples obtained at 100C and HOC developed slow moving bands of blotchy appearance which are characteristic for lambda-carrageenan fractions according to Hansen and Renoll (1974). The electrophoretic patterns of the individual 40C to 80C extracts, were complex and varied considerably between the different extraction samples, indicating the presence of mixtures of kappa, lambda, and possibly iota carrageenan. For extracts at 90C and above, the patterns were consistent with the electrophoretic behavior of relatively pure lambda Figure 15. Zonal Electrophoretic Patterns. 92 20 30 40 50 60 m ! 1 1 70 80 90 100 110 94 carrageenan. B. Molecular Weight and Size. The functional property of carrageenan stabilizer with respect to milk protein stabilization has been shown to be dependent on, among other factors, the molecular weight or molecular size(Chakraborty and Hansen, 1971; Vreeman et al., 1980). Several methods were used to estimate the molecular weight and size of the extraction samples obtained in this study, including gel permeation chromatography, analytical ultracentrifugation, intrinsic viscometry, and refractometry. 1• Gel Permeation Chromatography. Information related to the molecular weight distribution of a heterogeneous polymer can conveniently be obtained by gel permeation chromatography. Gel permeation chromatography of each extraction sample was conducted using a Sephadex 4B (cross linked) column (2.6 x 67cm) at 65C. The column was first calibrated with Dextran T molecular weight standards. The relationship established between the molecular weight and elution volume was Log(Mw)«-0.011537*(Elution Vol)+7.860839 (R**2R— .99987). The gel permeation (GP) chromatograms of the 95 carrageenan samples revealed a wide molecular weight distribution range from 11,000 to 3,370,000 daltons. The 40C extract(Fig. 16) showed quite homogeneous nature with peak heights around 330,000 daltons (elution volume 2 0 0 ml). The 70C extract(Fig. 17), was more heterogeneous than the 40C extract and had large portions of high molecular weight substances. The H O C extract(Fig. 18) was of exceptionally heterogeneous nature with several distinctive peaks showing. Considerable amounts of low molecular weight fractions (Mw<2 1 ,0 0 0 ) were noticed with this extract indicating that the heat degradation of the carrageenan polymer took place at this temperature of extraction. From the gel permeation chromatograms, the number and weight average molecular weights and inhomogeneity factor of each extract were calculated according to Vollmert (1973) (Table 12). The computer program used in the calculation is listd in Appendix A. The homogeneity of the molecular weight distribution pattern appeared well correlated with the numeric values for inhomogeneity (Table 12). Samples characterized by high values for inhomogeneity also showed multiple peaks. It appears that at low extraction temperatures(40-50C), carrageenans having average molecular weight (Mw) of around 550,000 daltons were first solubilized. A b the extraction temperature increased, the molecular weight of each extract RELATIVE CONC GO• 6 GC HOAORM F 0 EXTRACT 40C OF CHROMATOGRAM GPC . 16 . G I F O D I 0 13000 LTO VOL.,ML ELUTION VS EV VS CONC A 96 RELF1TIVE CONC. x GC HOAORM F 0 EXTRRCT 70C OF CHROMATOGRAM GPC . 7 1 . G I F =r ID “ C3 1 1 1 I ■ 1 I -j " I— " , !--j — r u i I J 1 I ■ ■ I "j 1 I 1 ! 1 J 0 10 6 IO 2 20 6 30 4 370 340 310 260 250 220 ISO 160 150100 LTO VOL.,ML ELUTION 0 0 4 97 RELATIVE CONC GC HOAORM F EXTRACi C O H OF CHROMATOGRAM GPC . 8 1 . G I F OJ “ ~ D I D LTO VOL.,ML , . L O V ELUTION S EV VS CONC ^ 98 99 Table 12. Dumber and Weight Average Molecular Weights of the Carrageenan Extraction Samples as Determined by Cel Permeation Chromatography. Extraction Average Molecular Weight T e m p .* C Mn Mw inhomogeneity(1) Factor 30 z9tj;ooci 586*000 0.96 40 314*000 531,000 0 . 6 8 50 260*000 586*000 1.25 60 118*000 692*000 4.86 70 391*000 782*000 1 . 0 0 80 2 0 0 * 0 0 0 652*000 2.26 90 181*000 581*000 2 . 2 1 1 0 0 60*000 492*000 7.18 1 1 0 77*000 520*000 5.84 1. Inhomogeneityc(Mw/Mn)-l. 100 became higher probably due to the increased quantity of lambda carrageenan which is characterized by a higher molecular weight than kappa. The previously reported values of kappa- and lambda-carregeenans are 296,000 and 761,000 daltons respectively (DeBai, 1979). However, the molecular weight of extracts above 70C did not increase significantly even though the concentration of lambda increased. This plateau in the molecular weight at high temperatures may be the result of a compensating influence of heat induced degradation. Masson(1954) has demonstrated in his heat degradation study of carrageenan that heat degradation * adversely affects the molecular weight of the carrageenan polymer at the temperatures above 60C where a first order random degradation takes place. 2. Molecular Weight Determination of Carrageenan Extraction Samples by Analytical Ultracentrifuqatlon. Analytical ultracentrifugation is a sensitive and reliable method for molecular weight determination of polymer materials and often used to obtain primary molecular weight values to which the corresponding values determined by other techniques can be calibrated. In this study, the high speed sedimentation equilibrium method (meniscus depletion) was used. The apparent weight averagee molecular weights, Mw(app), of each extract were 101 obtained at three different rotor speeds and at three different sample concentrations. The molecular weight calculation was aided by computer program(Appendix B), and the resulting values of Mw(app) are listed in Table 13. The obtained values of Mw(app) of the extract sample varied widely depending on the rotor speed and the concentration. As previously reported by Badui et al.(1978), Mw(app) of the carrageenan extraction samples increased as the rotor speed and /or the Bample concentration decreased. Due to the apparent pressure and concentration effects, a true molecular weight of this type of polymer must therefore be obtained by extrapolating the Mw(app) values to zero speed and zero concentration state. For that purpose the following multiple regression models with or without interaction terms were tested using SA5 program against the Mw(app) values to select the best fitting model for each extraction sample. Multiple Regression Models: Independent variables:Concentration(X), Speed(Y) Dependent variable {Apparent Molecular Weight(MW) 1 MW « a + b*X + c*Y 2 MW - a + b*X + c*Y + d*(X*Y) 102 Table 13. Apparent Weight Average Molecular Weights ,Mw(app), of the Carrageenan Extraction Samples. Extraction Rotor Apparent Weight Average Mol. Wt. Temp. Speed Concentration c RPM 0 .2 0 0 % 0.133% 0.067% 30 1 2 , 0 0 0 126,901 147,996 216,350 1 0 , 0 0 0 151,786 186,998 230,979 8 , 0 0 0 184,362 219,931 309,573 40 1 2 , 0 0 0 139,283 213,497 263,687 1 0 , 0 0 0 179,093 224,994 322,749 3,000 201,436 246,392 328,911 50 1 0 , 0 0 0 209,860 256,000 287,416 8 , 0 0 0 248,871 289,000 355,395 6 , 0 0 0 333,888 378,955 452,964 60 1 2 , 0 0 0 147,051 214,365 260,843 1 0 , 0 0 0 161,330 232,643 277,997 8 , 0 0 0 204,067 233,359 298,931 70 1 0 , 0 0 0 199,681 268,912 343,543 8 , 0 0 0 249,033 289,431 350,512 6 , 0 0 0 356,629 376,720 465,801 30 1 0 , 0 0 0 176,396 209,063 278,628 8 , 0 0 0 216,967 224,573 324,864 6 , 0 0 0 302,657 328,546 406,702 90 1 2 , 0 0 0 150,455 187,322 235,311 1 0 , 0 0 0 154,863 202,354 224,117 8 , 0 0 0 191,237 220,704 255,783 1 0 0 1 2 , 0 0 0 146,088 159,398 208,394 1 0 , 0 0 0 158,741 181,524 241,132 8 , 0 0 0 184,222 214,124 287,647 1 1 0 1 2 , 0 0 0 154,845 181,393 234,925 1 0 , 0 0 0 163,979 190,5B3 244,667 8 , 0 0 0 206,365 235,536 289,850 Seakem 2 14,000 134,941 140,801 163,234 1 2 , 0 0 0 153,071 171,223 195,838 1 0 , 0 0 0 175,018 198,133 240,148 103 3 MW - a + b*X + c*(Y**2) 4 MW - a + b*X + c*(X**2) 5 MW - a + b*X + c*Y + d*(X**2) 6 MW - a + b*X + c*Y + d*(Y**2) 7 MW - a + b*X + c*Y + d*(X**2) + e*(X*Y) a MW - a + b*Z + c*Y + d*(Y**2) + e*(X*Y) 9 MW - a + b*Z + c*Y + d*(X**2) e*(Y**2) 10 l/MW- a + b*X + c*Y 1 1 1/MW- a + b*X + c*Y + d*(X*Y) 12 1/MW- a + b*X + c*Y + d*(S**2) Selection of the best model was based on the criteria of the lowest possible t-test value and standard of error of estimate for the coefficients; a, b, c, d, and e. Table 14 lists the best model and the corrected (true) molecular weight, Mw(corr), for each carrageenan extract sample. The best models prevailing were Models 1 and 3 which indicates that the apparent molecular weight values obtained in this experiment were affected linearly by the concentration changes and either linearly or quadratically by the speed changes. The corrected molecular weight values did show wide variation between the different carrageenan extract samples; 400-500,000 daltons with low temperature extracts, larger molecular weight extracts at medium heat temperatures 104 Table 14. Corrected True Molecular Weight, Mw(corr), and Beet Fit Multiple Regression Model. Extraction Mvr Best fit multiple regression model Temp. C (corr) CONC SPEED SPEED**2 30 481,000 -735.76 -18.551 40 437,000 -990.88 - - 6 .7395*10**(-4) 50 554,000 -759.52 - -2.1089*10**(-3) 60(a) 383,000 -815.39 - -4.718*10**(-4) 70 767,000 -783.36 -44.589 - 80 628,000 -786.25 -31.151 - 90(b) 357,000 -563.27 -7.8863 - 1 0 0 424,000 -621.12 -14.343 - 1 1 0 360,000 -611.65 - -6.5243*10**(-4) Seakem 309,000 -341.06 ** -6.0273*10**(-4) a, bx unusual fringe patterns observed. 105 (around 70C), followed by smaller molecular weight extracts. In this respect the values agreed with the gel permeation chromatography data. In order to calibrate the OPC molecular weight values against the corresponding sedimentation equilibrium data, the two data sets were correlated. Generally the molecular weight values by GPC were much higher than their equivalents by sedimentation equilibrium. The obtained relationship was Mw(GPC)«0.4600Mw(corr) + 378215 with a correlation of about 0.70. Considerable discrepancy was observed with the 60C and 90C extracts which showed somewhat irregular distorted fringe patterns during the equilibrium run. When the two outlying values were excluded, the correlatin between the two different methods was improved (R**2 = 0.9605). 3. Intrinsic Viscosity. The hydrodynamic volumes of the polymers in the extract samples, were assessed by intrinsic viscosity measurements. Fig. 19 shows the viscosity values of each sample at varying concentrations. Intrinsic viscosity number of each sample obtained by extrapolating the viscosity values to zero concentration is listed in Table 15 and compared to the corresponding molecular weight values obtained by GPC and sedimentation equilibrium. The extract samples showed are higher in intrinsic viscosity values than Seakem 2, REDUCED VISCOSITY, DL/GM 0.0 FIGURE 19 . FIGURE . 02 0.3 0.2 0.1 NRNI VISCOSITY INTRINSIC OCNRTQ, 7. CONCENTRRTIQN, vs * w z X * EX + X50C EX X Y «> A X80C EX EX CONC SEAKEM EX EX X100C EX 60C EX 30C EX 70C 90C 40C 1 IOC 0.4 106 107 Table 15. Intrinsic Viscosity of the Carrageenan Extract and Corresponding Mvr by GPC and Mw(corr) by Sedimentation Equilibrium. Extraction 8 ed. Equilibrium del Permeation Intrinsic Viscosity T e m p ., C Mvr(corr) Mw dl/g 30 481,000 586,000 10.751 40 437,000 531,000 8 . 1 1 2 50 554,000 586,000 7.400 60 383,000 692,000 9.984 70 767,000 782,000 11.190 80 628,000 652,000 9.659 90 357,000 582,000 6.838 1 0 0 424,000 492,000 8.783 1 1 0 360,000 520,000 5.331 Seakem 2 309,000 2.626 108 indicating that the sequentral extraction was a far gentler treatment than the commercial process. The low viscosity value of Seakem 2 might be due to heat degradation in addition to some degree of alkaline degradation during the manufacturing process. 4. Refractive Index Determination. Refraction of light by a crystalline substance is largely dependent on the chemical composition, the molecular weight, and the molecular structure of the crystal. The refractive indices of the sample extract in the solid state and liquid state are tabulated in Table 16. Each extract under the polarizing microscope showed different degree of birefringence and was often observed as a mixture having more than one refractive index. Therefore, the reported values in Table 16 are average refractive indices. Considering that the threshold index difference of 0 . 0 0 2 is usually accepted in this type of measurement, the refractive index of each extract solid did not differ significantly. The same problem was encountered with the index value of the 0.4% solutions. Therefore, it was concluded that this approach was of no value for the study of polymer size or differentiation of species. However, it is possible that a refractometric evaluation of food Btabilizers may be useful for quality control purposes. 109 Table 16* Refractive Indices of the Extract Samples in Solid and Liquid State. Extraction Refractive Index (Ave.) Te m p ., C solids 0.4% solution(l) ...... 6 1 74S5~...... 1.3458 2 0 1.480 1.3451 30 1.465 1.3451 40 1.500 1.3454 50 1.493 1.3460 60 1.493 1.3458 70 1.496 1.3455 BO 1.500 1.3453 90 1.495 1.3450 1 0 0 1.493 1.3465 1 1 0 1.500 1.3450 1. Refractive index of 25C water was 1.3450 110 since significant differences between gums can be demonstrated. C. Stabilization of Alpha(Si)-Casein. Carrageenan possessess the ability to stabilize calcium sensitive milk proteins, in order to evaluate the specific functional property of each extract sample as a milk protein stabilizer, the following experiment was conducted with alpha(Si)-casein solution as a model system. The stabilization ability of each extract at four different carbohydrate/alpha(SI)-casein ratios was measured. The results are tabulated in Table 17. Statistical analysis of the stabilization test (Table 18) shows that casein stabilization was significantly different for the extracts and more significantly different for the carrageenan/casein ratios. Since the F ratios were significantly different, Duncan's multiple range test was conducted(Table 19). The results indicated that the carrageenan/casein ratios of 0.167 and 0.100 were significantly more effective than others. The carrageenan extracts obtained at 30, 40, and 50C stabilized the casein significantly better than the samples extracted at 60, 100, and HOC. Statistically, the Table 17. Stabilization of Alpha (SI)-Casein by the Extraction Samples. Extraction %Soluble Casein Carrageenan/casein ratio x 1 0 * * 2 T e m p .( c 0 . 0 3.3 1 0 . 0 16.7 30 4.65 31.27 76.87 79.33 40 4.65 38.89 69.38 73.39 50 4.09 39.64 68.67 59.97 60 4.65 16.28 41.73 35.40 70 5.45 29.55 51.36 58.28 BO 4.65 22.35 64.86 69.76 90 4.65 14.60 58.91 54.39 1 0 0 4.65 8.27 16.93 21.58 1 1 0 5.11 6.13 9.58 8.17 Seahem 4.65 8.50 38.24 67.18 112 Table Id. Analysis o£ Variance; Stabilization of Alpha(SI)-Casein by the Carrageenan Extraction Samples. Sources SSDF MS F F. 05 F.01 Total 24470 35 -- — Extract 6859 8 857 5.59** 2.36 3.36 Ratio 13931 3 4644 30.30** 3.30 4.72 Error 3670 24 153 ** Significant at both p<.05 and p<.01 levels. 113 Table 19. Duncan's Multiple Range Test: Stabilization of Alpha(SI)-Casein by the Carrageenan Extraction Samples. A. Carrageenan extract/casein ratio Grouping Mean N Ratio A 51.13 16.7 x 10(-2) A A 50.92 1 0 . 0 B 22.99 3.3 C 4.72 0 . 0 B. Carrageenan Extract Samples. Grouping Mean Extraction A 48.03 30 A A 46.58 40 A B A 43.09 50 B A B A 40.40 80 B A B A 36.14 70 B A B A 33.14 90 C 24.52 60 C 1 2 . 8 6 1 0 0 C 7.25 1 1 0 114 best casein stabilizer was the extract obtained at 30C followed by 40>50>80>70>90>60>100>110. Further evaluation of the stabilization data was conducted to compare physical and chemical properties of the extract samples. Correlations between the physical properties and casein stabilization at the two significantly different carrageenan/caseln ratios of 0.033 and 0.100 are shown in Table 20. The results show that the inhomogeneity factor with respect to molecular weight distribution is more important than the molecular weight itself. Stabilization appears to be highly affected by the overall chemical properties as the highest stabilization was with the most kappa(II)-rich extract and the lowest stabilization with the most lambda-rich fraction. Table 21 shows the effect of the molecular inhomogeneity on milk protein stabilization in comparison to the concentration of kappa. The results indicates that even though the concentration of kappa-fraction is significantly high in the stabilizer, the presence of polymers outside the effective molecular weight range may be not just ineffective but also detrimental to the stabilization of the milk protein. 115 Table 20. Correlations Between Physical, Chemical Parameters, and Casein Stabilization. Correlation Coefficients Ratio of Carrageenan Extract/casein Variables ______0.033 0.100______Physical Parameters Mw, OPC 0.474 0.360 MW, Sed. Eq. 0.236 0.279 Intrinsic Vise. 0.331 0.390 Re£. Index -0.170 -0.264 Molecular Inhomogeneity -O.905 -0.914 Chemical Parameters Kappa 0.860 0.795 iota -0.051 0.216 Kappa(II) 0.876 0.880 Lambda -0.876 -0.880 Kappa/Lambda 0.804 0.688 116 Table 21. The effect of molecular inhomogeneity on the milk protein stabilization. Extraction Molecular Stabilization ...... C Temp.# C Inhomoqeneity Alpha(SI)-casein Kappa 30 0.96 jsrsi 82.40 40 0 . 6 8 69.38 87.40 50 1.25 68.67 78.24 60 4.86 41.73 64.47 70 1 . 0 0 51.36 48.71 80 2.26 64.86 57.33 90 2 . 2 1 58.91 47.63 1 0 0 7.18 16.93 28.91 1 1 0 5.84 9.58 21.31 1. Carrageenan/casein ratio- 0.100(w/w). 2. Correlation coefficients: a. Stabilization/kappa: R**2- 0.880. b. Stabilization/lnhomogeneity: R**2- -0.914. DISCUSSION The current investigation has dealt with the physical and chemical characterization of carrageenan extracts obtained from Chondrus crispus by sequential extraction at gradually elevated temperatures from 5 to HOC. The purpose of this study has been to increase the understanding of the manner in which carrageenan fractions are released from the plant and, from this, to design an effective extraction method which may selectively yield highly functional carrageenan with respect to stabilization of fluid milk products. The major observations made in this study were: a. Distinctively different carrageenan compounds were sequentially released under the current extraction conditions. Cold extraction(5-20C) did not solubilize carrageean, but removed a number of soluble impurities including pigments and other low molecular substances. Kappa-carrageenan was concentrated in the low temperature extracts from 30-50C, followed by fractions rich in kappa and iota carrageenan at medium heat (60-80C); at high temperature (90-110C) lambda-rich fractions were 117 118 extracted* b. Each extract thus obtained showed significantly different ability to stabilize calcium sensitive proteins. Especially the low temperature fractions were superior stabilizing agents compared to the commercially extracted carrageenan stabilizer, Seakem 2. c. The molecular weight of each fraction showed strong temperature dependence. Heat degradation of the carrageenan extracts at high temperature was apparent; however, all the fractions showed higher molecular weights than the commercially extracted carrageenan stabilizer, Seakem 2* d. Molecular weight determination by analytical centrifugation showed def.inite non-ideal behavior since the apparent weight average molecular weights, Mw(app), of the carrageenan polymers were largely dependent upon the concentration and the rotor speed used. The application of multiple regression analysis'produced an empirical equation which permitted correction of the Mw(app) to conditions of zero concentration and zero speed. e. Refractive index measurements for assessments of molecular size of carrageenan was of no value in this study but the method may have application 119 in quality control in the stabilizer industry. A. The Mode of Sequential Carrageenan Extraction at Gradually Elevated Temperatures. As shown in Table 11# the mode of lambda release from ChondruB crispus was moderate with some sharp increase only at high extraction temperatures. On the other hand# as much as 70% of the total extractable kappa carrageenan in the plant had been released when the temperature reached 50C. The remaining kappa carrageenan was released at the higher temperatures along with iota and lambda carrageenan. These findings are in agreement with the results obtained by McCandless et al.(1976) in a study of the ultrasructural changes of Chondrus crispus during extraction in hot aqueous bicarbonate solution (Figs 3 and 4). The authors reached the conclusion that the microfibrilar components (basically kappa) are most readily disrupted by the extraction while intercellular granules (basically * lambda) are less dense after the extraction. They especially noticed that the most densely populated microfibrils in the outer edge of the cell walls resisted the effect of extraction more than the rest of the cell wall. The physical evidence provided by these workers suggests that the relative resistance of the carrageenan 120 substances in Chondrus crispus against heat disintegration during extraction could be in the increasing order of inner cell wall (kappa) < intercellular matrix (lambda) < outer cell wall (kappa). The stronger resistance of the outer cell wall, where the microfibrilar population is most dense, has not been fully explained in the study by McCandless et al.(1977). However, supposing that the native state kappa-carrageenan corresponds to a cation-associated gel (Gordon and McCandless, 1973; Chapman and Chapman, 1980), the disintegration of the outer cell wall microfibrils may well follow the sol-gel transition behavior of cation-induced carrageenan gel in vitro. The order-disorder transition of carrageenan in the gel form is highly concentration-dependent as observed by Rochas and Rinaudo(1980) who also reported that the increase of the transion temperature was far greater in the presence of counter ions. From this point of view, the strong resistance exhibited by the outer cell wall against extraction might possibly be due to the high density of carrageenan microfibrils in the particular outer cell wall. The sequence mode of carrageenan extraction from the seaweed shown in the presents study (Table 11) may be interpreted as followst The predominantly kappa type extracts at the low 121 extraction temperatures (30-50C) may correspond to the microfibrils in the less dense inner cell wall. At higher temperatures (60-110C), the more densely populated microfibrils in the outer cell wall may be solubilized due to the increased input of energy to disintegrate the carrageenan-cation aggregate (Rees, 1981). The gradual increase of the lambda type from the intercellular granules may be a consequence of the gradual decrease of the intercellular matrix density due to the continued extraction as well as to more complete disintegration of the intercellular granules facilitated by the high energy input at temperatures above 90C. B. The Physical and Functional Properties of the Sequential extracts• The present study showed that alpha(SI)-casein stabilization increased as the ratio of kappa to lambda increased, which is in good agreement with the findings of Hansen (1968), and Lin and Hansen (1970) who observed that the gel forming kappa was a more effective stabilizer than the non-gelling lambda. In addition to the conformation requirements of the structural sugar units with respect to gel-forming ability, a number of factors have been proposed for effective protein stabilization which include molecular size (Chakraborty and Hansen, 1971; Kardosh, 1975) and 122 molecular weight (Vreeman et al., 1980). In this study the effective molecular weight range for maximum stabilization was from 440,000 to 550,000 daltons for the kappa-rich extracts. Chakraborty and Hansen (1972) suggested that the the molecular weight range of the effective casein stabilizer lies between 100,000 and 300,000 daltons; however these values are based on Mw(app) and are, therefore, an underestimate. Molecular weight-dependence of stabilization of dairy products has later been reported by Vreeman et al., (1980) who observed that a minimum molecular weight of 1 0 0 , 0 0 0 daltons is the critical point for stabilization. Since the extracts examined in this study were not chemically purified nor alkali modified the stabilizing activity of each extraction fraction may involve the compensating effects between the chemical composition and the molecular weight and/or size. Molecular weight of each extract as determined by analytical ultracentrifugation showed that all of the extract samples including those exposed to possible heat degradation at 100 and HOC have considerably higher molecular weights than the commercially extracted Seakem 2. There is little doubt that the commercial process for carrageenan extraction involves much more severe heat treatment with risks for heat damage. Heat induced degradation was also evidenced by intrinsic viscosity of 123 Seakem 2 which showed a much smaller value than for any one of the extraction samples. The non-ideal behaviour of carrageenan in the equilibrium-sedimentation experiments is a subject which has been addressed by Desai (1979) and Badui et al., (1978). The present studies have confirmed that pressure and concentration effects are highly significant factors which must be taken into account whenever ultracentrifugation methods are used for molecular weight determination of carrageenan polymers. The application of impirical models based upon multiple regression analysis of all ultracentrifugation data have yielded corrected molecular weights in agreement with those obtained by Desai (1979). It was noteworty that the stabilization ability of each extract was significantly dependent on the homogeneity with respect to the molecular weight (Table 21). For example, the highly inhomogeneous 60C extract which contained 7.1% more kappa than the 80C extract showed 23.1% less stabilization power. The same comparison can be made with the 70 and 90C extracts which contained less kappa than the 60C extract but showed higher stabilization ability. The correlation coefficient between the kappa content of the fractions and their stabilization ability was + 0 .8 8 . However, the correlation between the molecular inhomogeneity 124 factor and stabilization was more pronounced with . R**2“-0.91. This finding may suggest that the presence of polymers outside the effective range does not add to stabilization effectiveness# but may well be detrimental to stabilization. It should be noted that the inhomogeneity factor for Seakem 2 and the associated stabilization effectiveness is consistent with this premise. Thus# the current study has produced convincing evidence that moderate temperatures for carrageenan extraction is essential for preservation of the carrageenan polymer and stabilization effectiveness. Furthermore# temperature programmed extraction may prove to be a highly effective method to obtain carrageenan stabilizers for specific uses. C. An Effective Method of Carrageenan Extraction. Although the physical and chemical properties of the extracts in this study have been characterized, it is possible that different results might have been obtained with different Chondrus crispus material due to the heterogeneous phycological properties of the seaweed which are affected by seasonal and geographical variation (Black et al.# 1965; Pickmere et al., 1975), and reproduction stage of life cycle (Taylor and Chen, 1973). 125 The ratio of kappa to lambda compounds of the extracts (Table 10) were different from that of Seakem 2 except for the 60C sample. The extracts obtained at temperatures below 60C showed a higher kappa content while the extracts above 60C contained more of the lambda fraction. Even though this indicates that the individual extracts obtained in this study possess clearly different chemical properties from the commercially extracted carrageenan, the overall ratio of the current extracts (Table 11) was 66.36% kappa to 33.64% lambda which is quite similar to that of Seakem 2 of 65.15% to 34.85%. What this is telling us is that the commercial extraction, characterized by a one-time cooking process at high temperature under slightly alkaline conditions, has produced a mixure of functional and non-functional carrageenans. For milk product stabilization, this may not only dilute the effectiveness but may also cause detrimental inhibition as was demonstrated by the inverse relationship between stabilization effectiveness and molecular inhomogeneity. The mode of extraction derived in this study suggests that the extractability of carrageenan compounds from the cellular matrix is largely dependent on the population density at the specific locations from the ultrastructural view. Also the degree of cation-specific aggregation of the gel forming carrageenans may control the rate of 126 solubilization to be extracted out. The following three-steps extraction is suggested as a le method for effective carrageenan extraction from Chondrus crispus. Step 1. Room temperature bleaching: This process will remove impurities including water soluble protein (enzyme), low molecular weight sugars and water soluble pigments. Step 2. Prolonged extraction at 50C: This fraction will represent kappa-rich extract yielding more than 50% of the total extractable carrageenan and 70% of the total extractable kappa-carrageenan and less than 30% of the total extractable lambda. Step 3. Extraction at 120C: This fraction will comprise 30% of the total extractable kappa carrageenan and 70% of the total extractable lambda part, which may be alkali converted to the gel-forming theta-carrageenan. Buffer condition: Aqueous alkaline buffer at pH 9.0 may be used in order to ease the extraction 127 by lowering the viscosity of the extractant and to protect the extractant carrageenan from heat degradation* SUMMARY Carrageenan polymers were sequentially extracted from Chondrus crispus at gradually elevated temperatures (5-110C) while maintaining the Ionic stregth at 0.5 and pH of the buffered solvent at 7.0. The resulting carrageenan extracts were characterized for their chemical, physical, and functional properties. It was observed that markedly different carrageenan polymers with respect to their content of kappa/iota/lambda fractions were released over the temperature range. Kappa-rich fractions were released from the plant at low extraction temperatures (30-50C). At medium temperatures (60-80C) the fractions were rich in kappa and iota carrageenans, and at high temperatures (90-110C) lambda-rich polymers were obtained. The sequential carrageenan extracts showed significantly different ability to stabilize alpha (SI)-casein against precipitation by calcium ions. Especially the low temperature fractions demonstrated superior stabilizing power compared to the commercially extracted carrageenan stabilizer Seakem 2. Molecular weight determination of the sequential extract by sedimentation equilibrium ultracentrigugation 128 129 (meniscus depletion method) showed highly non-ideal behavior o£ the polymers in which the apparent weight average molecular weight, Mw (app), was dependent upon the concentration and rotor speed. It was shown that Mw (app) values could be adjusted to zero concentration and zero speed through the use of an empirical equation of the form Mw (app) « Mw (corrected) + b*Speed + c*CONC in which the coefficients were determined by multiple regreggian analysis. The molecular weight of each fraction showed strong temperature dependence. Heat degradation of the carrageenan extracts at high temperature was apparent;however, all of the fractions showed higher molecular weights than the commercially extracted carrageenan stabilizer, Seakem 2. It was noted that the effectiveness of protein stabilization was inversely related to the molecular inhomogeneity factor calculated from gel filtration molecular weight distribution for each sample. The effective molecular weight range for maximum stabilization of the calcium sensitive alpha(Sl)-casein was from 440,000 to 550,000 daltons of the kappa-rich extracts. 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APPENDIX A COMPUTOR PROGRAM FOR MOLECULAR WEIGHT DETERMINATION BY GEL PERMEATION CHROMATOGRAPHY 1 PROGRAM 2 PRINT ******************************************************* * 3 PRINT •MOLECULAR WEIGHT DETERMNATION BY GEL CHROMATOGRAPHY' 4 PRINT • OF 5 PRINT ■ 40C EXTRACTION FRACTION' 7 PRINT '******************************************************* 8 PRINT ' ' 9 P R IN T ' KEY VARIABLES' 10 PRINT ' ' 11 P R IN T ' T: FRACTION TUBE NUMBER ' 1 2 P R IN T " EV! ELUTION VOLUME ■ 1 3 P R IN T ' b : a b s o r b a n c e o f b l a n k ' 1 4 P R IN T ' x: ABS OF SAMPLE ' 1 5 P R IN T " c: CORRECTED ABS AFTER BLANK* 1 6 P R IN T " Yi TOTAL OF CORR. ABS • 1 7 P R IN T ' p : p e r c e n t o f ' c o r r . a b s t o t o t a l • 1 8 B - * 1 19 ABS= ARRAY(41: > 20 LOADDATA (ABS»F40BC> 21 X=ABS/1000 22 PRINT ' ' 2 3 c=X-B 24 WHERE (X .LT. B) C=0 25 T=GRID(26r66j1) 2 6 EV= T*4 *77 2 7 Y=SUM COMPUTOR PROGRAM FOR MOLECULAR HEIGHT DETERMINATION BY SEDIMENTATION EQUILIBRIUM EDITING HODEL 1 . 0 0 PROGRAM 2 . 0 0 VBAR= . 5 3 0 3 . 0 0 DENSITY=1 . 0 1 4 7 1 1 6 4 . 0 0 TEMP=3 S 5 . 0 0 C0 NCS0 »2 0 0 *0 .1 3 3 *0 , 0 6 7 6 . 0 0 PRINT " • 7 . 0 0 PRINT "«#******************* * 1 0 . 3 0 PRINT '******#*******#**##** ' 1 0 . 6 0 PRINT 'RUN# 5 1 6 -3 » 1 4 0 0 0 RPM 1 0 . 9 0 RAl^ARRAYC1 0 .1 8 7 *1 0 .6 0 4 *1 0 .8 2 6 *1 0 ,9 7 7 *1 1 ,1 0 8 *1 1 .2 1 8 *1 1 .3 4 1 *1 1 . 4 4 6 ) 1 1 . 2 0 RA2 =ARRAYC 1 1 .5 2 8 *1 1 .6 0 8 *1 1 .7 0 6 *1 1 .7 8 7 *1 1 .8 6 4 *1 1 .9 4 0 *1 2 .0 0 7 *1 2 . 0 73) 1 1 . 5 0 R1=(RA1 *RA2 > 1 1 . 8 0 RB1»2 2 .1 7 0 *2 2 .6 0 3 *2 2 .8 1 1 *2 2 .9 6 2 *2 3 .0 8 6 *2 3 .1 7 9 *2 3 .2 8 6 *2 3 . 3 6 0 1 2 . 1 0 RB2 =2 3 .4 4 4 *2 3 .5 2 8 *2 3 .5 9 8 *2 3 .6 6 8 *2 3 .7 3 6 *2 3 . 8 0 3 1 2 . 4 0 R2 -(RB1 *RB2 > 12.70 RC1=34.392*34.723*34.890*35.040*35.166*35.240*35.309*35.377*35.4 4 9 1 3 . 0 0 RC2 S3 5 . 5 2 0 1 3 . 3 0 R3 =(RC1 *RC2 ) 1 3 . 6 0 FRG1 «GRID<1 *N0 ELS(R1 )*1 ) 13.90 FRG2=GRID<1*N0ELS ***#*ttt*#****##**t## RUN* 516-3, 14000RPM RUN*516-2, SEAKEM, 12000RPH RUN#516-1, SEAKEM, 10000RPM FIRST RUN DATA ANALYSYS J 12,000RPM • WIRE MENIS1 MENIS2 MENIS3 OUTEDGE HAGFACT FACTOR <«« ****** <««« <««« ««<« ««<« ****** . 0 0 2 7 . 0 0 3 1 8 . 4 7 4 3 0 .307 37.123 22.096 ; FRINGERDOUT LNFREALR RSQ HALFRSQ ««« < « « « « « » « ******* ******* ******* 1 1 0 . 1 8 7 0 6 . 0 8 1 3 6 . 9 7 9 1 8 . 4 9 2 1 0 . 6 0 4 • 6 9 3 1 5 6 . 0 9 9 9 37.209 18.604 3 1 0 . 8 2 6 1.0986 6.11 37.332 1 8 . 6 6 6 4 1 0 . 9 7 7 1 . 3 8 6 3 6 . 1 1 6 8 3 7 . 4 1 5 1 8 . 7 0 8 5 1 1 . 1 0 8 1 . 6 0 9 4 6.1227 37.488 1 8 . 7 4 4 6 11.218 1.7918 6 . 1 2 7 7 3 7 . 5 4 9 1 8 . 7 7 4 7 11.341 1.9459 6 * 1 3 3 3 3 7 . 6 1 7 1 8 . 8 0 8 8 11.446 2.0794 6 . 1 3 8 3 7 . 6 7 5 1 8 . 8 3 8 9 1 1 . 5 2 8 2 . 1 9 7 2 6.1417 37.721 1 8 . 8 6 1 0 1 1 . 6 0 8 2 . 3 0 2 6 6.1453 37.765 18.883 11 1 1 . 7 0 6 2 . 3 9 7 9 6.1498 37.82 1 8 . 9 1 12 11.787 2.4849 6*1534 37.865 1 8 . 9 3 2 1 3 1 1 . 8 6 4 2 . 5 6 4 9 6.1569 37.908 1 8 . 9 5 4 14 1 1 . 9 4 2.6391 6.1604 37.95 18.975 1 5 1 2 . 0 0 7 2 . 7 0 8 1 6.1634 37.988 1 8 . 9 9 4 16 12.073 2.7726 6.1664 38.024 19.012 (tire) (ml)