Effect of feeding protected cholesterol on ruminant milk fat secretion

Feeding 1–2 g/day of cholesterol protected against ruminal hydrogenation caused a 20–30% drop in the secretion of milk fat by goats and cows. The effect was observed with goats fed conventional rations or with goats and cows fed rations supplemented with protected lipids, but was not observed with cows fed conventional rations, or when unprotected cholesterol and protected β-sitosterol was fed to these animals. The results suggest that this depression in milk fat is due to a decreased uptake of plasma triacylglycerol fatty acids by the mammary gland, induced by dietary cholesterol.     

Effect of 7-methylated bile acids and bile alcohols on cholesterol metabolism in hamsters

The effect of 7-methyl substituted bile acid and bile alcohol analogues on cholesterol metabolism was studied in the hamster. Animals were fed chow plus 0.1% cholesterol supplemented with 0.1% of one of the following steroids: chenodeoxycholic acid, 7-methyl-chenodeoxy-cholic acid, 7β-methyl-24-nor-5β-cholestane-3α,7α,25-triol, cholic acid, 7-methyl-cholic acid, or 7β-methyl-24-nor-5β-cholestane-3α,7α,12α,25-tetrol. Cholesterol absorption was determined from fecal analysis after feeding of radiolabeled cholesterol and β-sitosterol. Of the six compounds studied, chenodeoxycholic acid and 7-methyl-chenodeoxycholic acid decreased intestinal cholesterol absorption (17% and 31% decrease, respectively). Only 7-methyl-chenodeoxycholic acid decreased serum cholesterol concentration (29% decrease), but there were no analogous changes of liver and biliary cholesterol concentration and cholesterol saturation of bile. Total fecal neutral sterol excretion was increased in the groups fed chenodeoxycholic acid and 7-methyl-chenodeoxycholic acid. In addition, the production of coprostanol was increased in both groups. These data suggest that 7-methyl-chenodeoxycholic acid resembles chenodeoxycholic acid in its effect on cholesterol metabolism and may be a potential candidate for further studies of its gallstone-dissolving properties.     

Palmitic acid enhances cholesterol gallstone incidence in sasco hamsters fed cholesterol enriched diets

In an established hamster model of cholesterol cholelithiasis, a semipurified lithogenic diet containing 4% butterfat and 0.3% cholesterol leads to the production of cholesterol gallstones in only 50–60% of animals after a 6-wk feeding period. The purpose of this study was to investigate whether gallstone incidence could be increased while feeding a nutritionally adequate diet of moderate cholesterol content. The semipurified lithogenic diet was modified as follows: (i) substitution of 1.2% palmitic acid for 4% butterfat, and (ii) varying the amount of dietary cholesterol from 0.0 to 0.3% with either butterfat or palmitic acid as the lipid component of the diet. Substitution of palmitic acid for butterfat produced a significantly higher incidence of cholesterol gallstones (94%vs. 53%). Palmitic acid also raised the incidence of gallstones when added to the 0.1% and 0.2% cholesterol diets as compared to butterfat: 0%vs. 44% and 50%vs. 81%, respectively. Gallstone incidence increased from 0% to nearly 100% when the cholesterol content of the palmitic acid diets was raised from 0.0% to 0.3%, indicating a dose response effect with respect to dietary cholesterol. Hamsters fed cholesterol-free diets did not form gallstones. Increased dietary cholesterol led to increased liver weight associated with a significant increase in liver cholesterol concentration. However, the palmitic acid groups had significantly lower liver cholesterol values than the corresponding butterfat groups. Serum and biliary cholesterol concentrations increased with increasing dietary cholesterol intake, but there were no differences between the butterfat and palmitic acid groups. The cholesterol saturation index increased from 0.56 to 1.32 in the butterfat groups and from 0.56 to 1.30 in the palmitic acid groups upon raising the dietary cholesterol from 0.0 to 0.3%. Biliary total bile acid concentration did not vary significantly within all groups; however, the addition of cholesterol produced an increase in the ratio of chenodeoxycholic acid to cholic acid. It is concluded that in Sasco hamsters the saturated fatty acid, palmitic acid, when substituted for butterfat in a nutritionally adequate lithogenic diet, is capable of increasing gallstone incidence to almost 100% during a 6-wk feeding period.     

Oxidative interactions of cholesterol in the milk fat globule membrane

The effects of oxidative interactions between cholesterol and milk fat globule membrane (MFGM) components,i.e., nonlipid fraction, total lipid, nonpolar lipid and polar lipid, on cholesterol oxidation were studied in the presence and absence of water. In the dry state, cholesterol natively present in MFGM appeared to be protected at 135°C. The nonpolar lipid and nonlipid fraction contributed to the protective effect of MFGM. Added cholesterol accelerated the oxidation of membrane lipid fractions. At 75°C, pure cholesterol and membrane lipid fractions did not show significant interaction. However, cholesterol and other lipids in MFGM were less stable than when these were heated separately. When cholesterol and membrane lipids were mixed in an aqueous medium at 75°C, each accelerated the oxidation of the other. The MFGM exhibited a high protective effect on cholesterol oxidation in an aqueous environment. The nonlipid fraction protected cholesterol against oxidation, whereas the lipid fraction was destructive. In the absence of water, the net balance between these two opposing factors was destructive. The presence of water reversed the balance in favor of protection. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

The effect of alfalfa-corn diets on cholesterol metabolism and gallstones in prairie dogs

Cholesterol gallstones were present in prairie dogs fed alfalfa plus corn with and without exogenous cholesterol (0.4%). The diets fed to the animals for eight weeks contained alfalfa plus corn in fixed proportions of 50∶50, 85∶15 and 15∶85 (w/w). At sacrifice, all animals were healthy but had not gained weight; no deaths occurred during the experiment. Cholesterol gallstones were present in all groups. In the absence of exogenous cholesterol, the highest stone incidence was found in the animals which received the lowest fiber (highest corn) diets (alfalfa plus corn, 50∶50, 67%; alfalfa plus corn, 15∶85, 83%). Cholesterol gallstone incidence was 100% when exogenous cholesterol was added to the alfalfa plus corn diets (50∶50 and 15∶85). No pigment gallstones were detected in any animal. Liver and plasma cholesterol concentrations were highest in the animals receiving alfalfa plus corn (15∶85) plus 0.4% cholesterol (4.29 mg/g, and 356 mg/dl, respectively). These values were lowest in animals receiving 85% alfalfa plus 15% corn without cholesterol (2.19 mg/g and 88 mg/dl, respectively). Lithogenic indices were below 1.00 in all groups. Biliary bile acids were mainly amidates of cholic acid and chenodeoxycholic acid, with the former predominating. Thus, gallstones can be formed in prairie dogs in the absence of exogenous cholesterol; gallstone incidence is reduced by dietary fiber.     

Effect of milk fat fractions on fat bloom in dark chocolate

Anhydrous milk fat was dissolved in acetone (1∶4 wt/vol) and progressively fractionated at 5°C increments from 25 to 0°C. Six solid fractions and one 0°C liquid fraction were obtained. Melting point, melting profile, solid fat content (SFC), fatty acid and triglyceride profiles were measured for each milk fat fraction (MFF). In general, there was a trend of decreased melting point, melting profile, SFC, long-chain saturated fatty acids and large acyl carbonnumbered triglycerides with decreasing fractionation temperature. The MFFs were then added to dark chocolate at 2% (w/w) addition level. In addition, two control chocolates were made, one with 2% (w/w) full milk fat and the other with 2% (w/w) additional cocoa butter. The chocolate samples were evaluated for degree of temper, hardness and fat bloom. Fat bloom was induced with continuous temperature cycling between 26.7 and 15.7°C at 6-h intervals and monitored with a colorimeter. Chocolate hardness results showed softer chocolates with the 10°C solid fraction and low-melting fractions, and harder chocolates with high-melting fractions. Accelerated bloom tests indicated that the 10°C solid MFF and higher-melting fractions (25 to 15°C solid fractions) inhibited bloom, while the lowermelting MFFs (5 and 0°C solid fractions and 0°C liquid fraction) induced bloom compared to the control chocolates.     

Dietary oxidized cholesterol modulates cholesterol metabolism and linoleic acid desaturation in rats fed high-cholesterol diets

The interactive effect of high dietary levels of oxidized cholesterol on exogenous cholerterol and linoleic acid metabolism was examined in male 4-wk-old Sprague-Dawley rats given high-cholesterol diets. The rats were pair-fed purified diets free of or containing either 0.5% cholesterol alone or both 0.5% cholesterol and 0.5% oxidized cholesterol mixture (containing 93% oxidized cholesterol) for 3 wk. Hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity was reduced in rats given cholesterol alone or both cholesterol and oxidized cholesterol. However, hepatic cholesterol 7α-hydroxylase activity was lowered only when rats were given both cholesterol and oxidized cholesterol, although dietary cholesterol increased this activity. Reflecting this effect, acidic steroid excretion was lowest among the groups of rats given cholesterol and oxidized cholesterol. On the other hand, the activity of hepatic Δ6 desaturase, a key enzyme in the metabolism of linoleic acid to arachidonic acid, was increased in rats given both cholesterol and oxidized cholesterol, although dietary cholesterol alone lowered its activity. As a result, the Δ6 desaturation index, 20∶3n-6+20∶4n-6/18∶2n-6, in liver and serum phosphlipids tended to be higher in the group fed both cholesterol and oxidized cholesterol than in the one fed cholesterol alone. Thus, dietary oxidized cholesterol significantly modulated exogenous cholesterol metabolism and promoted linoleic acid desaturation even when it was given at high levels together with a high cholesterol diet.     

Effects of diets high in saturated fat and cholesterol on the lipid composition of canine platelets

The phospholipid composition of platelets from dogs on various experimental diets was determined. Thyroidectomized foxhounds were fed a control diet or the control diet supplemented with (1) beef tallow, (2) beef tallow and cholesterol, or (3) beef tallow, cholesterol, and safflower oil for 23 weeks prior to isolation of platelets. Platelets from animals fed the control diet contained 36.7% phosphatidylcholine (PC), 22.8% phosphatidylethanolamine (PE), 18.4% sphingomyelin (Sph), 11.8% phosphatidylserine (PS), 6.3% phosphatidylinositol (PI), and 2.2% lysophosphatidylcholine. The PE was 77.6% in the plasmalogen form. No highly significant changes in the phospholipid class composition resulted from the experimental diets. Cholesterol supplementation of the diets, however, caused consistent alterations in the fatty acid compositions of the platelet phospholipids including increases in the percentages of 18∶1ω9 (oleic acid), 18∶2ω6 (linoleic acid), and 20∶3ω6 (homo-gamma linolenic acid) and a decrease in the percentage of 20∶4ω6 (arachidonic acid). Addition of safflower oil to the tallow-cholesterol diet partially reversed these effects. These cholesterol-induced alterations in fatty acid composition could be due to exchange with plasma lipids, de novo synthesis, or altered platelet metabolism. The mechanism remains to be determined. Der. Nelson’s current affiliation is the Lipid Metabolism Branch, Division of Heart and Vascular Diseases, National Heart, Lung, and Blood Institute.     

Effect of DAG on milk fat TAG crystallization

The effect of milk fat and standard DAG on the crystallization behavior of milk fat TAG (MF-TAG) was investigated. When milk fat DAG were added to MF-TAG at the 0.1 wt% level, crystallization was delayed. Racemic purity was shown to be an important factor in the ability of DAG to influence TAG crystallization. Only sn-1,2 isomers of blends of MF-TAG with 0.1 wt% of the racemic mixtures of dipalmitin and diolein increased the activation free energy barrier to MF-TAG nucleation (ΔG _c ) and delayed the subsequent crystallization process by increasing the crystallization induction time (τ_SFC) determined from solid fat content-time measurements. Although crystallization kinetics were affected, the properties of the resulting network structures remained unchanged upon addition of milk fat minor components at the 0.1 wt% level     

Acetoacetate metabolism of rats fed high fat or restricted calorie diets

Ethyl-¹⁴C-acetoacetate was used to trace oxidation and metabolism of acetoacetate when rats were fed a high fat diet (80% of total calories from beef tallow or corn oil, carbohydrate free), a high carbohydrate diet (2% corn oil) or a high carbohydrate diet with restriction of calories to one half of ad lib. consumption for two weeks. The rate of expiration of¹⁴CO₂ in all groups of animals did not differ significantly and was not related to plasma concentration of acetoacetate. The high fat diets slightly enhanced the oxidation of acetoacetate to¹⁴CO₂ over a 3 hr period compared to other diets. Incorporation of acetoacetate into fatty acids did not differ significantly among groups. Rats fed the high carbohydrate diet ad lib. incorporated into liver cholesterol more acetoacetate than did any other group, but dietary unsaturated fat resulted in greater incorporation of acetoacetate into cholesterol than saturated fat. High calorie and high beef tallow groups were ketonemic but the low concentration of plasma acetoacetate in rats fed a high corn oil diet indicates that unsaturated fatty acids are not ketogenic. The data show that utilization of acetoacetate is not significantly reduced in a ketonemic condition and support the premise that overproduction of ketone bodies is the cause of ketonemia. Rats appeared to be normal during the two-week period when no carbohydrate was included in the diet. Presented at the AOCS Meeting, Chicago, October, 1967.     

Response of plasma lipids to dietary cholesterol and wine polyphenols in rats fed polyunsaturated fat diets

This experiment was designed to evaluate the effects of dietary red wine phenolic compounds (WP) and cholesterol on lipid oxidation and transport in rats. For 5 wk, weanling rats were fed polyunsaturated fat diets (n−6/n−3=6.4) supplemented or not supplemented with either 3 g/kg diet of cholesterol, 5 g/kg diet of WP, or both. The concentrations of triacylglycerols (TAG, P<0.01) and cholesterol (P<0.0002) were reduced in fasting plasma of rats fed cholesterol despite the cholesterol enrichment of very low density lipoprotein + low density lipoprotein (VLDL+LDL). The response was due to the much lower plasma concentration of high density lipoprotein (HDL) (−35%, P<0.0001). In contrast, TAG and cholesteryl ester (CE) accumulated in liver (+120 and +450%, respectively, P<0.0001). However, the cholesterol content of liver microsomes was not affected. Dietary cholesterol altered the distribution of fatty acids mainly by reducing the ratio of arachidonic acid to linoleic acid (P<0.0001) in plasma VLDL+LDL (−35%) and HDL (−42%) and in liver TAG (−42%), CE (−78%), and phospholipids (−28%). Dietary WP had little or no effect on these variables. On the other hand, dietary cholesterol lowered the α-tocopherol concentration in VLDL+LDL (−40%, P<0.003) and in microsomes (−60%, P<0.0001). In contrast, dietary WP increased the concentration in microsomes (+21%, P<0.0001), but had no effect on the concentration in VLDL+LDL. Cholesterol feeding decreased (P<0.006) whereas WP feeding increased (P<0.0001) the resistance of VLDL+LDL to copper-induced oxidation. The production of conjugated dienes after 25 h of oxidation ranged between 650 (WP without cholesterol) and 2,560 (cholesterol without WP) μmol/g VLDL+LDL protein. These findings show that dietary WP were absorbed at sufficient levels to contribute to the protection of polyunsaturated fatty acids in plasma and membranes. They could also reduce the consumption of α-tocopherol and endogenous antioxidants. The responses suggest that, in humans, these substances may be beneficial by reducing the deleterious effects of a dietary overload of cholesterol.     

Effect of phospholipids on isothermal crystallisation and fractionation of milk fat

Milk fats with different concentrations of water and phospholipids (PL) were crystallised isothermally under static conditions and their crystallisation behaviour was monitored by Differential Scanning calorimetry (DSC) and pulsed nuclear magnetic resonance (pNMR). The Avrami and the Gompertz models, which were fitted by non‐linear regression, described the crystallisation process. A significant effect of phospholipid concentration was observed using both techniques (DSC and pNMR). Especially the induction time and the Avrami growth rate constant were altered: higher amounts of PL delayed the onset of static crystallisation. A similar effect of PL on the crystallisation kinetics was observed in a small‐scale fractionation. Moreover, the filtration time of the crystal suspension and melting properties of the stearin were strongly affected by the presence of higher concentrations of PL. These observations emphasise the importance of the adequate removal of PL during anhydrous milk fat production.     

Effect of processing conditions on crystallization kinetics of a milk fat model system

Crystallization behavior of three blends of 30, 40, and 50% of high-melting fraction (MDP=47.5°C) in low-melting fraction (MDP=16.5°C) of milk fat was studied under dynamic conditions in laboratory scale. The effect of cooling and agitation rates, crystallization temperature, and chemical composition of the blends on the morphology, crystal size distribution, crystal thermal behavior, polymorphism, and crystalline chemical composition was investigated by light microscopy, differential scanning calorimetry (DSC), X-ray diffraction (XRD) and gas chromatography (GC). Different nucleation and growth behavior were found for different cooling rates. At slow cooling rate, larger crystals were formed, whereas at fast cooling rate, smaller crystals appeared together. Slowly crystallized samples had a broader distribution of crystal size. Crystallization temperatures had a similar effect as cooling rate. At higher crystallization temperatures, larger crystals and a broader crystal size distribution were found. Agitation rate had a marked effect on crystal size. Higher agitation rates lead to smaller crystal size. Cooling rate was the most influential parameter in crystal thermal behavior and composition. Slowly crystallized samples showed a broader melting diagram and an enrichment of long-chain triacylglycerols. Crystallization behavior was more related to processing conditions than to chemical composition of blends.     

Effect of cupric ions on serum and liver cholesterol metabolism

Cupric ions were administered subcutaneously to male Sprague-Dawley rats at a single dose of 200 μmol/kg. At 24 hr after administration, a remarkable increase of total and free cholesterol was seen in the rat serum. Also, when lecithin-cholesterol acyltransferase (LCAT) (E.C. 2.3.1.43) activity was expressed as the percentage of the total serum that free cholesterol esterified, the acyltransferase activity in rats treated with cupric ions showed a slight decrease while the triglyceride content in rat serum and liver decreased by 54% and 61%, respectively. However, the content of hepatic cholesterol in rats treated with cupric ions did not show such a marked change. On the other hand, acid cholesteryl ester hydrolase activity (Acid CEH) (E.C. 3.1.1.14) in liver lysosomes of rats treated with cupric ions showed a marked decrease with increasing cupric ion concentration both in vivo and in vitro. Furthermore, cupric ions caused a marked release of the lysosomal enzymes cathepsin D and β-glucuronidase into the cytosolic fraction. The changes in acid cholesteryl ester hydrolase activity induced by cupric ions appear to be a direct effect of cupric ions on the enzyme. These results suggest that excessive cupric ion concentrations could cause various disorders in lipid metabolism.     

Effect of glucose administration on cholesterol and bile acid metabolism in rats

Glucose administered to fasted rats caused a marked stimulation in hepatic cholesterogenesis and cholesterol 7 alpha-hydroxylation, and an increase in biliary excretion of cholesterol and total bile acids. The excretion of cholic acid was not incluenced during the first few hr after glucose administration, but was significantly increased after 5 hr. Chenodeoxycholic acid showed a similar change, but the increase was only ca. one tenth of that of cholic acid. The excretion of deoxycholic acid was markedly increased by 1 hr, but gradually decreased thereafter. Pretreatment with neomycin abolished the increase in deoxycholic acid by fasting and glucose administration. Other bile acid components showed no significant change. It thus was presumed that cholesterol endogenously synthesized in the liver was metabolized mainly to cholic acid. In contrast, exogenous cholesterol was metabolized mainly to chenodeoxycholic acid. During the period of the acute enhancement of cholic acid formation from the endogenous cholesterol, biliary excretion of deoxycholic acid was increased. This probably occurred through the depression of 7 alpha-rehydroxylation of deoxycholic acid, or through the enhancement of microbial formation of deoxycholic acid in the lumen, and through the increase of intestinal absorption.     

Effect of candicidin on cholesterol and bile acid metabolism in the rat

Sterol metabolism studies were carried out in rats maintained on a diet containing a polyene antibiotic, candicidin, (30 mg/kg/day) for 2-1/2 months. Compared to the controls, the candicidintreated animals had a smaller food intake and weight gain during this period. There was no difference between the 2 groups in serum cholesterol levels, biliary cholesterol or bile acid concentrations. However, in the experimental group, liver cholesterol content decreased by 27% and hepatic HMG-CoA reductase increased by 36%. Candicidin administration produced an 84% increase in neutral sterol output without change in bile acid output. Cholesterol absorption was reduced 80% by candicidin feeding. The weight of ventral prostate was reduced 33% by candicidin administration. Prostatic HMG-CoA reductase levels were 3 times higher than those of the liver, but enzyme activity was unchanged by candicidin treatment.     

CLA isomers in milk fat from cows fed diets with high levels of unsaturated fatty acids

The concentrations of CLA isomers were determined by Ag⁺-HPLC in the milk fat of cows fed a control diet consisting of hay ad libitum and 15 kg of fodder beets or this diet supplemented with oilseeds containing either high levels of oleic acid (rapeseed), linoleic acid (sunflower seed), or α-linolenic acid (linseed). Highly significant (P≤0.001) correlations were found between the daily intakes of oleic acid and the concentration of the CLA isomer trans-7,cis-9 in milk fat; of linoleic acid and the CLA isomers trans-10,trans-12, trans-9,trans-11, trans-8,trans-10, trans-7,trans-9, trans-10,cis-12, cis-9,trans-11, trans-8,cis-10, and trans-7,cis-9; and of α-linolenic acid and the CLA isomers trans-12,trans-14, trans-11,trans-13, cis,trans/trans,cis-12,14, trans-11,cis-13, and cis-11,trans-13. CLA concentrations were also determined in the milk fat of cows grazing in the lowlands (600–650 m), the mountains (900–1210 m), and the highlands (1275–2120 m). The concentrations of many isomers were highest in milk fat from the highlands, but only three CLA isomers (cis-9,trans-11, trans-11,cis-13, and trans-8,cis-10) showed a nearly linear increase with elevation. Therefore, these three CLA isomers, and particularly the CLA isomer trans-11,cis-13, the second-most important CLA in milk fat from cows grazing at the three altitudes, could be useful indicators of milk products of Alpine origin.     

Effect of struture and form on the ability of plant sterols to inhibit cholesterol absorption in hamsters

We investigated the effect of three types of plant sterols (4-desmethylsterols, 4,4′-dimethylsterols, and pentacyclic triterpene alcohols) in three forms (free, esterified with FA, or with phenolic acids) on cholesterol absorption. Plant sterol fractions derived from soybean (99% 4-desmethylsterols), rice bran (70% 4,4′-dimethylsterols), or shea nut (89% pentacyclic triterpene alcohols) were fed to male hamsters (n=20/group) as free sterols or esterified with FA or phenolic acids (cinnamic or ferulic). Cholesterol absorption was measured after 5–8.5 (mean, 7) wk by a dual-isotope technique. Soybean sterol intake significantly reduced cholesterol absorption efficiency (23%) and plasma total cholesterol (11%). Rice bran sterols tended to lower cholesterol absorption efficiency by 7% and plasma total cholesterol by 5%, whereas shea nut sterols had no effect. In hamsters, dietary 4-desmethylsterols were more effective than 4,4′-dimethylsterols in lowering cholesterol absorption and levels of cholesterol in blood. Pentacyclic triterpene alcohols had no effect on the absorption of cholesterol or on its level in blood. Esterification with FA did not impair the ability of 4-desmethylsterols and 4,4′-dimethylsterols to inhibit cholesterol absorption, whereas esterification with phenolic acids reduced this ability. This study supports the use of 4-desmethylsterols, esterified with FA to increase solubility, as the most effective cholesterol-lowering plant sterols in the diet.     

Effect of processing conditions on physical properties of a milk fat model system: Microstructure

The effect of processing conditions on the microstructure of three blends of 30, 40, and 50% high-melting fraction [Mettler dropping point (MDP)=47.5°C] in the lowmelting fraction (MDP=16.5°C) of milk fat was studied. The effect of cooling and agitation rates, crystallization temperature, chemical composition of the blends, and storage time on crystalline microstructure (number, size, distribution, etc.) was investigated by confocal laser scanning microscopy (CLSM). To improve resolution, a mix of Nile blue and Nile red dyes was dissolved in the melted samples in proportions that did not modify the nucleation kinetics. Samples were then crystallized by cooling (0.2 or 5.5°C/min) to crystallization temperature (25, 27.5, and 30°C). After 2 h at crystallization temperature, a slurry was placed on a microscope slide and samples were stored 24 h at 10°C. During this period, more material crystallized. Slowly crystallized samples (0.2°C/min) formed different structures from rapidly crystallized samples (5.3°C/min). Crystals were sometimes diffuse and hard to distinguish from the liquid. Samples were darker as a result of this solid-mass distribution. However, rapidly crystallized samples had well-defined crystals and seemed to be separated by a distinct liquid phase. These crystals were not in touch with each other as was the case for slowly crystallized samples. Higher agitation rates led to smaller crystal size due to enhanced nucleation. Larger crystals were formed when crystallization occurred at higher temperatures. Storage time resulted in an increase of crystal size. Larger crystal size and structures with more evident links had a more elastic behavior with higher elastic modulus E’.