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Fisheries Science 65(5), 742-749 (1999)

Composition and Rheological Properties of Extracellular Mucilage from Marine Bacterium Klebsiella oxytoca CF154

Fu-Jin Wang,*1 Chorng-Liang Pan,*2 and Ching-Shyong WU*2,•õ *1Department of Food Science , China College of Marine Technology and Commerce, 212 Yeng-Ping North Road, Taipei, Taiwan I11, R.O.C. *2Department of Food Science , National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, Taiwan 202, R.O.C. (Received July 17, 1998)

Lyophilized extracellular mucilage (LEM) powder was recovered from Klebsiella oxytoca CF154 culture, and the yield was approximately 0.8 g per liter of the culture medium. Relative viscosity (RV) in creased as the concentration of LEM aqueous solution increased from 0.0 to 0.5%. Factors affecting RV of 0.2% LEM solution showed that the RV was stable over a pH range of 4 to 9, although it dropped sharply outside of this range. The higher NaCl concentration (0-5%) in LEM solutions was, the lower RV of LEM solutions was. RV of 0.2% LEM solution decreased as the temperature increased from 10 to 80•Ž. Changes in molecular flexibility, which was indicated as changes in activation energy of 0.2% LEM solution, depended on temperature. Results from Sephacryl S-400 gel permeation chro matography (GPC) indicate that LEM is a mixture of protein- complexes of which the dominant fraction (85%) has a molecular weight ranging from 1.5-1.8 MDa in the polysaccharide moie ty. HPLC analysis suggested that the major in this dominant fraction were , , , and . In addition, there exists uronic acid at 6.3% in this dominant fraction. Key words: Klebsiella, polysaccharide, relative viscosity, extracellular mucilage (EM)

Many microorganisms produce exopolymers that pro which produces a yellowish, round, and mucoid colony. It mote adhesion to other cells or to inert surfaces.1) These ex can use or sugar alcohol such as , , opolymers are mostly long-chain which , glucose, mannose, raffinose, adonitol, mannitol, dissolve or disperse in water to give a thickening or viscosi or sorbitol as sole carbon source. Glucose is fermented by ty-building effect.2-6) Some microbial exopolymers have K. oxytoca CF 154 with the production of acid and gas (H2, been successfully used in the food, textile, pharmaceutical, or H2S). Optimal temperature for growth of this strain is agricultural, paint, and petroleum industries during the 25 to 27•Ž.14) The extracellular mucilage was extracted and last three decades.7-8) Among them, from isolated from K. oxytoca CF154.151 The present study aims Xanthomonas campestris,8) gellan gum from Pseudomo to investigate the physicochemical properties of the ex nas elodea and Sphingomonas paucimobilis,8-10) pullulan tracellular mucilage from K. oxytoca CF154 and to evalu from A ureobasidium pullulans and Kluyveromyces fragi ate the possibility of its application to food and other uses lis,8.11)and curdlan from Alcaligenes faecalis var. myx in the future. ogenes 1OC38,12)are now used commercially as gelatiniz ers, stabilizers, thickeners, or plastic materials in the food Materials and Methods industry.8,10,12)While from Leuconostoc mesen teroides has also been commercially exploited for use as a Bacterial Strain and Storage Conditions blood expander,8) now it might be suitable for use as a Klebsiella oxytoca CF 154 was isolated from the biofoul source of , as a cryostabilizer in frozen food material on a fishing boat surface.14) The strain was routine systems, and as a low calorie bulking agent for ly cultured in marine broth 2216 (MB; Difco, Detroit, sweeteners.13) USA) at 26•Ž. Stock cultures were made by mixing a pure The sources of commercial microbial exopolysaccha culture that had been grown with an equal volume of a rides are exclusively soil microorganisms.6,8) Knowledge of 100% glycerol (v/v) and the mixture was stored at the exopolysaccharides produced by marine is still -70•Ž . lacking although this area is worth studying .1,4,11) in our

previous studies 14,15) Klebsiella oxytoca CF154, a bacteri Medium and Growth Conditions um which produces capsules of a mucous substance, was Active cultures were obtained by inoculating 1 loop of isolated from the biofoul material on the hull of a fishing -70•Ž stock culture into 50 ml of fresh MB . The broth boat. The strain CF154 is a gram-negative, nonmotile, was then incubated at 26•Ž by shaking at 150 rpm for 20 catalase (+), and short rod (1.78 ƒÊm •~ 0.66ƒÊm) aerobe h. Active culture samples of K. oxytoca CFI 54 (1 ml;

•õ Corresponding author: Wu, Ching-Shyong, Ph.D., Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan 202, R.O.C. Extracellular Mucilage from K. oxvtoca CF154 743 about 107-108 CFU / ml, colony forming unit per milliliter) at 480 nm) according to Dubois et al.17)Protein content in were put into 250 ml Erlenmeyer flasks containing 100 ml each fraction was also calculated by determining the absor of MSBB (mucus screening basal broth).14) The MSBB bance at 280 nm.18) RV of the fractions was also ascer medium comprised 0.5% (w/v) polypeptone (BBL, Cock tained, and only the fractions with an RV greater than or

ysville, USA), 0.1% yeast extract and 5% in 1 I ar equal to 1.5 as well as with the absorbance at 280 nm and tificial sea water.16) The inoculated MSBB was incubated at 480 nm greater than 0.1 were pooled together and used in 26•Ž by shaking at 150 rpm for 52 h using modified the following procedure. These fractions were dialyzed method of Liang et al.14) After 52 h incubation, most of and lyophilized to give white powders. These powders the exopolysaccharide accumulation occured during the in were called refined LEM, and its molecular weight was de itial stationary phase of growth and the bacterial cells of termined graphically by plotting the log of the molecular the culture reached 108-109 CFU / ml. weight of the dextran standard against the elution volume.

Preparation of Lyophilized Extracellular Mucilage (LEM) Proximate Composition Analysis Powder Total sugars in the crude EM, LEM, and refined LEM Following 52 h incubation, cultures of strain CF154 powder were determined using the phenol-sulfuric acid were centrifuged at 17,600 •~ g (Hitachi, Himac CR21, method.17) Standard methods recommended by the Tokyy, Japan) fr 30 min at 4•Ž to get the sediment of the AOAC19) were used to determine crude protein (micro-Kjel bacterial cells. Pellets were collected and then resuspended dahl method), moisture, and ash contents in these three in 100 ml distilled water. To increase extractability of EM, EM powders. Each determination was replicated five the pellet resuspesion was heated at 80•Ž for 60 min in a times. water bath using modified method of Pan and Liang.15) Af ter heat treatment, the bacterial cells were examined by a Solubility transmission electron microscope (Jeol EX2000, Tokyo, LEM powder (0.1 g, dry mass) was dissolved in 10 ml of Japan). The heat-treated solution was centrifuged at the following different solvents, (1) distilled water (pH 17,600 •~ g for 20 min at 10•Ž to remove the bacteria. 6.58), (2) 1-5% (w/v) NaCl (pH 6.64), (3) 0.1-0.2% (w/v) Ethanol was added to and mixed with the supernatant to NaOH (pH 10.32-11.10) or 35.0% (v/v) ammonia water give a ethanol final concentration of 71.25% (v/v) and was (pH 9.87), (4) 80.0% (v/v) formic acid (pH 4.01), (5) let stand for 6 h at 4•Ž. Flocculent aggregates were ob 99.0% (v/ v) dimethyl sulphoxide (DMSO), 99.0% (v/ v) served occasionally on the top or in the upper part of this methyl alcohol, 95.0% (v/v) ethyl alcohol, 95.0% (v/v) mixed solution. After being collected by centrifugation at propyl alcohol, 99.5% (v/v) acetone, 90.0% (v/v) ether, 17,600 •~ g for 30 min at 15 •Ž, the aggregates were washed or 95.0% propylene glycol. This 1% (w/v) LEM solution twice with 95% ethanol at 15•Ž. The final aggregates was prepared and stirred for 5 min to dissolve and/or dis (17,600)•~g, 30 min) were resuspended at 1% (w/v) in perse at 25 •} 1 •Ž. The 1 % (w / v) LEM solution was cen deionized water and lyophilized, and this was designated trifuged at 28,000 x g (Hitachi, Himac CR21, Tokyo, as crude extracellular mucilage (crude EM) powder. The Japan) for 30 min at 25•Ž to collect supernatant, and the final aggregates (17,600 •~ g, 30 min) were resuspended at dried LEM (dry mass) in the supernatant was determined. 1% (w/v) in deionized water and then dialyzed (molecular The solubility (% sol) is calculated as the ratio of the weight cutoff size 1 •~ 104 Da) in running deionized water amount of LEM in supernatant (g, dry mass) to the origi for 24 h at 4•Ž. Finally, the dialyzed solution was lyophi nal amount of LEM in dispersion (0.1 g) according to lized, and this was designated as lyophilized extracellular Regenstein and Regenstein.20) The 1 % LEM solution was mucilage (LEM) powder. categorized as soluble (solubility> 80%), partially soluble (20% < solubility < 80%), or insoluble (solubility < 20%) in Analysis of LEM Powder each solvent tested. LEM powder was dissolved in deionized water (25 •} VC, pH 6.58) to form the stock of 0.2% (w/v) aqueous so Viscosity lution, which was used in the following analytical proce Relative viscosity (RV) of the stock LEM solution was dure except solubility experiment. measured with a capillary viscometer, Cannon-Fenske No. 100 (Cannon, Tamson, Holland) in a water bath at Gel Permeation Chromatography 20•}0.5•Ž. Deionized water was used as a reference. RV Gel permeation chromatography (GPC) was performed was calculated as the ratio of the flow time of the sample on a Sephacryl S-400 HR (Pharmacia, Uppsala, Sweden) to that of the reference. Factors that were expected to column (XK 26 mm/ 100 cm) with 0.1 M phosphate buffer affect the RV of the LEM solution, including NaCl concen solution (pH 7.0) as an eluting solution. The column was tration (1-5%), temperature (10-70•Ž), and pH (1-12), calibrated by using a mixture of dextran standard (Pharma were investigated. Following Launay et al.21) intrinsic vis cia, Uppsala, Sweden) with molecular weights of 712 kDa cosity (ƒÅ) was defined as equal to iƒÅred(reduced viscosity) and 2,000 kDa at a concentration of 0.25 mg/ml. A sam when sample concentration approached zero; ƒÅred is equal ple (1.0 ml) of the 0.2% stock LEM solution was applied to 77, (specific viscosity) divided by solute concentration to a column and eluted with 0.1 M phosphate buffer at a (C); 11,p is equal to RV minus 1. That is: ƒÅ=(ƒÅred)c•¨o, where flow rate of 1.2 ml/ min. The eluate was collected every 6 ƒÅrea=RV-1/C. m1in a fraction, A hundred fractions were collected. Total sugar content of each fraction was determined by the Analytical Methods for Polysaccharide of Refined LEM phenol-sulfuric acid method (determining the absorbance High performance liquid chromatography One gram of 744 Wang et al . refined LEM was deproteinized twice by the Sevag method 22) and then hydrolyzed in 1 N sulfuric acid at 100•Ž for 4 h. The hydrolyzate was neutralized with barium car bonate, and the filtrate was passed through a column pack ed with Dowex-1 resin (OH form, 50-100 mesh, Sigma, St. Louis, USA) and a column packed with Dowex 50W (H+ form, 100-200 mesh, Sigma, St. Louis, USA) using the modified methods of Yang et al.23) The neutral sugars were eluted with deionized water, concentrated under reduced pressure and then examined by high performance liquid chromatography (HPLC, Tosoh PX8010, Tokyo, Japan) with a refractive index detector (Gilson, Middle ton, USA) and integrator D-2500 (LDC Analytical Co., Riviera Beach, USA). The size of the column was 300 mm (length) •~ 7.8 mm (I. D.), and it was packed with LiChrosorb NH2 5•}m (Phenomenex, Torrance, USA). The column was eluted with the mixture of acetonitrile and deionized water in the ratio of 80:20 (v/v) with a flow rate of 0.5 ml/min at 90•Ž. Uronic acid and pyruvic acid of 0.2% refined LEM, elut ed from a column packed with Amberlite resin IRA-420 (Sigma, St. Louis, USA) with 0.5 N sodium hydroxide af ter removing neutral sugars, were determined according to Wardi et al.24) and Berntsson,25) respectively. Sulfate con tent of refined LEM was also measured following the method of Terho and Hartiala.26)

Results and Discussions

LEM Extraction and Yield from Broth Pan and Liang15) concluded that heat extraction in a water bath at 75•Ž for 60 min gave the highest relative vis cosity (RV) of the crude extracellular mucilage (EM) from K. oxytoca CF154. In order to investigate the best extractability of EM, K. oxytoca CF154, culture was heat treated in 75•Ž, 80•Ž and 90•Ž water bath for 60 min in the preliminary study. Transmission electron micrograph was used to examine the release of EM from cell after heat treatment. The strain CF154 was disrupted after heat treat ment with 90•Ž water for 60 min, while the whole cell membrane was still maintained after heat treatment at 75•Ž and 80•Ž for 60 min. In this study, K. oxytoca CF154 culture was placed in an 80•Ž water bath for 20, 40, 60, and 80 min. Transmission electron micrograph was also used to examine the release of EM from cells during Fig. 1. Transmission electron micrographs of K. oxytoca CF 154 during heat treatment, and the results are shown in Fig. 1. In the heat treatment at 80•Ž. early stages of heating for 20 min, the capsule structure of (A) After heating for 20 min, the capsules become loosen. (B) Af K. oxytoca CF154 became loosen state and partially ter heating for 40 min, aggregates are visible around the cell. (C) Af detached from the cell envelope, and bubbles were also ob ter heating for 60 min, the cells are almost completely nude. served in this capsule (Fig. 1A). As the heating continued, minute aggregates began to form possibly due to the denaturation of protein moieties, which in turn weakened in the cell wall was found under this heat treatment. When the attachment between the capsule layer and the cell wall the heating time was prolonged to 80 min, the bacterial cell (Fig. 1B). After 60 min, the capsule layer was completely was disrupted (data not shown). dissolved and the almost intact cell wall boundary of the The yield of LEM powder prepared from one liter of nude cell could be clearly seen (Fig. 1C). Absorbance at bacterial culture was approximately 0.75-0.85 g. This is an 260 nm and 280 nm in extractive solution before and after order of magnitude less than 6.5 g, the yield of highly vis heat treatment at 80•Ž for 60 min were measured. There cous exopolysaccharide accumulated per liter of culture of was no difference before and after the treatment in absor Sphingomonas paucimobilis, a soil strain.10) A new bacteri bance at 260 nm and 280 nm, which were 1.34 and 1.65, re um isolated from river water, Klebsiella sp. produces the spectively. So this dissolution of the capsule layer might be extracellular polysaccharide BS-1,4) of which yield has useful in further purification processes because no leakage reached 12.8 g/1.271The yield of LEM powder in this study Extracellular Mucilage from K. oxytoca CF154 745 can thus be seen to be rather low. MSBB might not be the cohol, glycerin, and propylene alcohol, but incompatible optimal medium for producing LEM, and we are now en with detergents.29) is insoluble in water, alkali gaged in trying to increase the yield of LEM from K. oxyto and organic solvents but soluble in many dilute aqueous or ca CF 154 culture by adjusting the medium and growth con ganic acids at concentrations in the range of 0.25 to 10% ditions. (at pH levels below 6). These acids includes formic, acetic,

propionic, oxalic, malonic, succinic, lactic, pyruvic, malic Characteristcs of LEM and Refined LEM Powder and citric acids.301 Gellan gum needs to be heated to dis Chemical composition of LEM powder As shown Table solve and requires cation to bring about gelation as the 1, the content of total sugar, uronic acid, and crude pro solution cools.7,9) Curdlan is a water-soluble acidic polysac tein of LEM are significantly higher than those of crude charide, and it forms a high-set gel when its aqueous sus EM but ash amount of LEM is lower than that of crude pension is heated to about 70-100•Ž.7,12) Pullulan is also a EM. This result is considered to be due to the effect of dial water-soluble polysaccharide, and it is also compatible ysis. Ash was retained at only 3% in LEM after dialysis. with water-soluble polymers, such as gelatin, polyvinyl al After being recovered from gel permeation chromato cohol, and .11)

graph, the contents of total sugar and uronic acid of refined LEM are higher than those of LEM. And the Viscosity amounts of ash and crude protein of refined LEM are sig LEM powders were collected and resolved in deionized nificantly lower than that of LEM. This suggests that some water (25 •} VC, pH 6.58) at a concentration of 0.2% (w / low molecular weight components were removed by GPC. v) homogeneously, then the 0.2% (w/v) LEM solution Solubility of LEM Powder While the concentration of was used for the following experiments. LEM in deionized water (pH 6.58) was lower than 0.6% Effects of LEM concentration and NaCl solution on rela tive viscosity The effect of different concentrations of (w/v) at temperature from 10•Ž to 80•Ž, the LEM solu tion existed as a soluble and homogeneous suspension. NaCl on relative viscosity (RV) of different concentrations However, when the concentration of LEM in water was of LEM solution are shown in Fig. 2. The RV of LEM solu higher than 0.6% (w/v), its solution was viscous, opaque tions increased with increasing LEM concentration from and heterogeneous in the same temperature range. At 0.1% to 0.5%, but the RV of the LEM solutions were 50•Ž, 1% LEM powder was soluble in deionized water, as reduced when NaCl concentrations were increased. Table well as hydrotropic salt solution (1-5% NaCI solution, pH 2 shows that the intrinsic viscosity of LEM solution 6.64), producing a viscous opaque solution. The solubility decreased with increasing NaCl concentration from 0 to characteristics of LEM powder were similar to those of 5%. One possible explanation for these observations is another microbial gum, xanthan.z$1 The solubility proper that the hydrodynamic volume of LEM, which depends ties of LEM suggest that it can be used as a thickener in the food industry. 1% LEM powder was also soluble in 0.1 1.2 N sodium and potassium hydroxide, 80.0% formic acid, and 35.0% ammonium hydroxide, while insoluble in 99.0% methanol, 95.0% ethanol, 95.0% isopropanol, 99.5% acetone, and 90.0% ethyl ether. In addition, 1% LEM powder was also soluble in ethyl alcohol and propy lene glycol as their concentrations were lower than 20% and 30%, respectively. Aqueous solutions of xanthan gum will be tolerant of certain hydrophilic solvents such as ethanol and propylene glycol up to 50%.zB1 All carragee nans are soluble in hot water and hot milk, but insoluble in organic solvents. Carrageenans are compatible with al

Table 1. Quantitative analysis of crude EM, LEM and refined LEM powders from Klebsiella oxytoca CF154

Fig. 2. Effect of LEM concentration on relative viscosity of aqueous so lution with different NaC1 concentrations. NaCl concentrations: •œ: 0%; •¡: 1%; •£: 3%; •¥: 5%.

Table 2. Intrinsic viscosity of LEM solution with different NaC1 concentrations

*1 Total sugar was analyzed by phenol-sulfuric acid method.17) Uronic acid, pyru vate, sulfate, and protein contents were determined by Harmine sulfuric acid calorimetric method,24) salicylaldehyde colorimetric method,25) sodium rhodizonate colorimetric method,26) and AOAC method,18) respectively. *1 Intrinsic viscosity of LEM solution was calculated according to Launay et al.20) *2 ND: not detected. 746 Wang et al.

primarily on molecular weight, chain rigidity, and solvent From the calculation according to the Arrhenius equation, quality can begin to overlap each other at higher concentra the value of EQof LEM aqueous solution is lower than 335 tions.20) Therefore, the RV decreased when the KJ/g.mole (the average molecular weight of LEM was hydrodynamic volume reduced. So, the higher the concen 1.65 MDa per mol and which was used to estimate Eo of tration of NaCI is, the less space is available for the LEM LEM aqueous solution according to the Arrhenius equa molecule to expand. This is probably because the presence tion). LEM aqueous solution has a lower EQ than typical of counter ions (cations) in an LEM solution lowers the stiff polysaccharides such as xanthan gum (Es: 1900 RV of the solution by reducing electrostatic repulsion 1100),34) diacetate (E.: 645),351and (EQ: among the anionic groups such as uronic acid (see Table 1) 2100-2520).36 In all, the molecule of LEM aqueous solu on the LEM molecule chain. Similar results were also ob tion seems to behave softer than that of polysaccharides served in carrageenan solution29) and in xanthan gum solu mentioned above such as xanthan gum and pectin. tion.28) In these cases, too, the decrease in intrinsic viscosi ty with the increase of ionic strength was apparently due to Determination of Molecular Weight and Sugar Compo neutralization by the counter ions of Nay. As we propose nents of Refined LEM in the present case, neutralization probably reduces the in When gel permeation chromatography (GPC) was used tramolecular electrostatic repulsive force, and thereby the to analyze the 0.2% LEM solution, the fraction numbers degree of molecular unfolding decreases and consequently from 25 to 45 produced an absorbance peak at both of 280 intrinsic viscosity lowers .28,29) and 480 nm, which represent the content of protein and Effects of pH and temperature on relative viscosity Figure 3 shows that the relative viscosity of 0.2% LEM so lution remained relatively unchanged from pH 4 to 9. This stability of viscosity over a wide pH range is similar to the stability of viscosity found in the succinoglycan from Al caligenes faecalis var. myxogenes. The viscosity was con stant from pH 3 to 10.12)A similar pattern was observed in xanthan gum, PS3a24, and PS3a25.2) The relative viscosity of 0.2% LEM solution decreased with increasing temperature in the range of 10 to 70•Ž as shown in Fig. 4A. Filar and Wrick30) observed the similar temperature effect on chitosan solution between 25 and 60•Ž. Figure 4B shows the linear relationship between log [RV] and the inverse of the absolute temperature [I/ T(x 10-3 K-1)] in the temperature range of 20-60•Ž. The slope of the linear regression equation in Fig. 4B is propor tional to the flow activation energy of LEM aqueous solution as calculated from the Arrhenius equation.31) Niel sen32) and Chen and Lin33) proposed that the value of the activation energy (En) for the flow process is related to the chain flexibility of the molecule. The molecules of polysac charide in the aqueous solution with a higher chain flexibili ty have a lower activation energy for the flow process .32,33)

Fig. 4. Viscosity of 0.2% LEM aqueous solution (pH 7.0) at different temperatures. (A) plot of relative viscosity versus temperature, (B) plot of Log (relative viscosity) versus inverse of absolute temperature (T-1, K-1 •~ 10-3). Fig. 3. Relative viscosity of 0.2% LEM aqueous solution at different The regression curve of (B) was obtained as r=0.9437, pH. y=0.64096+ 386.6358X. Extracellular Mucilage from K. oxytoca CF154 747

Fig. 5. Gel permeation chromatograms of (A) 0.2% LEM aqueous solution and (B) mucus screening basal medium (MSBB) by monitoring the con tent of protein (A280nm, left) and total sugar (A480, right) at 25•Ž.

total sugar, respectively (Fig. 5A). These fractions were also the ones that showed a peak of RV (data not shown). Figure 5B shows the protein and sugar content of mucus screening basal medium (MSBB) before fermentation. It was obviously observed that the protein and sugar in MSBB were distributed between fraction numbers 50 and 80 by GPC analyses. Thus fraction numbers 25 to 45 large ly account for the observed viscosity of the LEM solution. These eluate fractions presumably contain complexes of polysaccharide and protein compounds. The refined LEM, extracted from these elutes, was calculated to have an average molecular weight of approximately 1.5-1.8 MDa (Fig. 6). This result is comparable to the results for well known bacterial polysaccharides such as xanthan gum.36) The HPLC analysis of the hydrolyzed refined LEM rev ealed that the major sugar components of the refined LEM were mannose, galactose, glucose, and arabinose in the ra tio of 13.2:5.0:1.9:1.0 (data represent the mean of three Fig. 6. GPC pattern of refined LEM and calibration curve for determi replications, Table 3). When carboxyl-reduction of the nation of its molecular weight. refined LEM was not executed, the uronic acid content of The molecular weight of dextran standards are 2,000 kDa and 712 0.2% refined LEM was determined about 6.1% (w/w), kDa in spot A and B, respectively. but pyruvic acid group was not detected (Table 1). Ex opolysaccharide was anionic in nature due to the presence of uronic acids. Uronic acids have also been reported to be components of xanthan gum , alginic acid, gellan, and h yaluronic acid.2,38) In addition, pyruvic acid has been reported to be a significant component of xanthan gum and other marine microbial exopolysaccharide PS 3a35.2,38,39) l Calculated from the HPLC analysis of hydrolyzed LEM Many microbial exopolysaccharides contain pyruvic . acid linked acetalically to 0-4 and 0-6 of a ƒÀ3-D-galac

topyranosyl , ƒÀ3-D-glucopyranosyl, ƒÀ3-D-mannopyranosyl, or 2-acetamido-2-deoxy-D-glucopyranosyl residue. Fur might therefore be an acidic heteropolysaccharide. While thermore , pyruvic acid might play in important role on the the sugar composition of the refined LEM was very differ viscosities property of mucous solutions .3) Refined LEM ent from that found in other Klebsiella sp.,40,41) the BS-1

* 748 Wang et al, produced by Klebsiella from river water was also an acidic 1821 (1988). heteropolysaccharide composed of galactose and glucuron 5) C. S. Buller and K. C. Voepel: Production and purification of an ex ic acid in the molar ratio of 4:1,40) Sutherland7) proposed tracellular polyglucan produced by Cellomonas flavigena strain that the sugar composition of Klebsiella sp. most common KU. J. Indust. Microbiol., 5, 139-146 (1990). ly comprises up to three neutral hexasaccharides (includ 6) V. Crescenzi: Microbial polysaccharides of applied interest: On ing 6-deoxysugars) and a uronic acid, forming repeat units going research activities in Europe. Biotechnol. Prog., 11, 251-259 (1995). of 3-5 sugars. Many serotypes form groups of closely relat 7) 1. W. Sutherland: Structure-function relationships in microbial ex ed structures, in which the difference may be as small as opolysaccharides. Adv. Microbiol. Physiol., 12, 393-448 (1994). the presence or absence of an acetyl group.7) Heidelberger 8) J. D. Dziezak: A focus on gums. Food Technol., 45, 116-120 (1991). and Nimmich41) reported that ten polysaccharides of Kleb 9) D. Lobas, S. Schumpe, and W.-D. Deckwer: The production of gel siella contained galactose, glucuronic acid and glucose or Ian exopolysaccharide with Spingomonas paucimobilis E2 mannose, and , and five of them contained (DSM6314). Appl. Microbiol. Biotech., 37, 411-415 (1992). 10) A. A. Ashtaputre and A. K. Shah: Studies on a viscous, gel-form pyruvic acid. The differences in chemical composition and ing exopolysaccharide from Sphingomonas paucimobilis GSI. structure of these polysaccharides from Klebsiella may be Appl. Environ. Microbiol., 61, 1159-1162 (1995). related to their immunological specificity. 41) 11) Y. C. Shin, Y. H. Kim, H. S. Lee, and S. J. Cho: Production of ex According to the above results, LEM powder presents opolysaccharide pullulan from by a mixed culture of Aureo viscosities at low concentrations but is not like xanthan basidium pullulans and Kluyveromyces fragilis. Biotechnol. Bioen gin., 33, 129-133 (1989). gum, which has uniform viscosity over the temperature 12) T. Harada, M. Terasaki, and A. Harada: Microbial polysaccha range of 0-80•Ž. 0.2% LEM solution also has solubility rides, in: "Industrial Gums" (ed. by R. L. Whistler and J. N. de and moderate stability in the pH range of 4.0 to 8.0. So it Miller), Academic Press, New York, 1993, pp. 427-445. seems to be useful for quality improvement as a thickener. 13) D. D. McCurdy, H. D. Goff, D. W. Stanley, and A. P. Stone: Rheo In addition, when we discussed with Prof. Jenn-Shou Tsai logical properties of dextran related to food applications. Food in Dept. of Food Science of NTOU (National Taiwan Oce Hydrocolloids, 8, 609-623 (1994). 14) J.-F. Liang, C.-L. Pan, and F.-J. Wang: Screening of mucoid bac an University), he pointed out that the emulsifying proper terial strains from fishing boat surface. Food Sci, (in Chinese), 22, ties of EM examined in his lab showed a good potential for 86-98 (1995). the future utilization in the food system (personal com 15) C.-L. Pan and J.-F. Liang: The separation of mucous compound munication). The EM tested was extracted from the MSBB from Klebsiella oxytoca CF154 and the preliminary study on the culture of K. oxytoca CF154 at 60, 80, or 100•Ž for 60 min nitrogen and carbon sources of the medium. Food Sci. (in Chinese), and then lyophilized and named as EM60, EM80, and 22, 99-112 (1995). EM100, respectively. The emulsifying activity and emul 16) B. Austin, D. A. Allen, A. Zachary, M. R. Belas, and R. R. Colwell: Ecology and taxonomy of bacteria attaching to wood sur sion stability of EM100 had the best effect and followed by faces in a tropical harbor. Can. J. Microbiol., 30, 447-461 (1979). EM80 and was EM60 at the concentration of 1% (w/v). 17) M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. The emulsifying activity and emulsion stability of 1 % Smith: Colorimetric method for determination of sugars and relat EM100 were the same as those of commercial emulsifiers ed substances. Anal. Chem., 28, 350-354 (1956). such as sugar ester, Tween 80, xanthan gum, and arabic 18) H. Wu: A new colorimetric method for the determination of plas gum at the same concentration. The film-forming ability ma proteins. J. Biol. Chem., 51, 33-39 (1922). of LEM as well as the improvement effect of LEM on gel 19) AOAC: Official Methods of Analysis, 15th ed. (ed. by Association of Official Analytical Chemists), Washington, D. C., 1990. forming property of mackerel surimi are also being stud 20) J. M. Regenstein and C. E. Regenstein: Protein functionality for ied currently. Extensive studies on LEM powder have been food scientists, in "Food Protein Chemistry" (ed. by J. 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