Biotechnol. Rog. 1992, 8, 327-334
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An Anionic Galactomannan Polysaccharide Gum from a Newly-Isolated Lactose-Utilizing Bacterium. I. Strain Description and Gum Characterization James H. Flatt,+Robert S. Hardin> Juan M. Gonzalez,s Dina E. Dogger, Edwin N. Lightfoot, and Douglas C.Cameron* Department of Chemical Engineering, University of Wisconsin, 1415 Johnson Drive, Madison, Wisconsin 53706-1691
As part of an effort to obtain microorganisms able to produce polysaccharide gums from whey and whey permeate, soil samples from farm fields regularly treated with whey were screened for bacteria able to produce gums from lactose. The most promising organism isolated (ATCC 55046) is a facultative anaerobe, tentatively identified as a new Erwinia species on the basis of biochemical and morphological testa. The organism produces a polysaccharide gum from lactose and other sugars (herein named lactan gum) composed of mannose, galactose, and galacturonic acid with an approximate molar ratio of 53:2 and containing no organic acid modifying groups. The weight average molecular weight of the gum is approximately 7 X lo6. Aqueous solutions of lactan gum exhibit shear-thinning and elastic flow behavior with an estimated power law model flow index of 0.26 a t 1% (w/w) gum. The viscosity of aqueous 1% (w/w) lactan gum solutions is stable over a p H range of 2-11, being particularly stable in alkaline environments. Aqueous 1% (w/w) gum solutions a t p H 5-11 show excellent thermostability, retaining a t least 80% of the original viscosity after being heated to 121 OC for 15 min. These flow properties indicate potential industrial applications in food and nonfood products requiring a moderate degree of thickening, wet-end additives and coating agents for paper products, ceramics, detergents, and binders for building materials.
Introduction The problem of whey utilization has plagued the dairy industry for the past several decades and is worsening as the worldwide demand for cheese products increases. More than 25% of all milk produced in the U.S. is converted to cheese. Nine kilograms of whey is produced as a byproduct for every 1 kg of cheese produced. Ultrafiltration processing of whey enables the removal of valuable proteins from whey, but it does little to utilize the majority of solids which remain in the permeate stream. Whey permeate (partiallydeproteinized whey) is a dilute solution of lactose (4.4-4.9%),protein(0.8%),ash(0.5-0.7%),andacid (0.20.5%) (Coton, 1980;Morr, 1984). Lactose is the primary solid component of whey and is responsible for its high biological oxygen demand (BOD) of approximately 50 OW/ L. Approximately 40 billion kg annually, roughly 475% of the total worldwide whey production, is waste and is disposed of in waste treatment facilities or on farm fields at a significant cost to the dairy industry (Clark, 1987; Hoogstraten, 1987). Although some attempts at whey utilization have been successful, approximately 537% of the whey in the U.S. remains unutilized (Clark,1987;Morr, 1984). The primary limitation of most proposed processes for whey utilization has been the lack of economic feasibility, rather than the lack of technical feasibility (Flatt, 1990). We have investigated the production of microbial polysaccharide gums from lactose as part of our effort to
* Author to whom correspondence should be addressed.
+ Current address: Kelco, Division of Merck, San Diego, CA 92113. Current address: Advanced Tissue Sciences, LaJolla, CA 92037.
t 8
Current address: Pillsbury, St. Louis Park, MN 55416.
identify and develop economically feasible processes for the utilization of lactose in whey and whey permeate. Polysaccharidegums are attractive products from lactose because (1) they can be produced efficiently on substrates with relatively low sugar concentrations and high carbon to nitrogen ratios such as whey and whey permeate, (2) they are intermediate value specialty chemicals with sufficient value to offset part of the costs of whey transportation and concentration, and (3) an internal market for gums exists in the dairy industry for products such as cream cheese and ice cream. Polysaccharidegums are valuable to industry primarily due to their ability to modify the rheology of aqueous systems. Gums are useful in the food industry as thickening, suspending, emulsifying, stabilizing, lubricating, film-forming, water-retaining, and chelating agents. In nonfood industries, they are used in adhesives, pastes, building materials, cleaners, polishes, seed coatings, binders, paper products, petroleum and water-well drilling muds, enhanced oil recovery, wastewater treatment, cosmetics, and pharmaceuticals (Pace, 1987;Wells, 1977). In this paper, we describe a newly isolated lactoseutilizing soil bacterium and the properties of the polysaccharide gum (which we have named lactan gum) produced by this organism. The fermentationkinetics and metabolic aspects of polysaccharidegum production by this organism are presented in the following paper (Flatt et al., 1992).
Materials and Methods Strain Isolation. Soil samples from farm fields regularlytreated with whey and whey permeate were taken from locations within Wisconsin. Each sample was enriched for lactose-utilizingmicroorganismsby culturing
8756-7938/92/3008-0327$03.00/0 0 1992 American Chemical Society and American Institute of Chemical Engineers
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roughly 2 g of soil in 100mL of lactose semidefined isolation medium (LSIM),consisting of the following (in grams per liter): lactose monohydrate, 5; (NH4)&304,0.16; KzHPO4, 3.6; KHzP04, 1.8; MgS04.7Hz0, 0.6; CaC1~2H20,0.04; FeS04.7Hz0,0.0019; CoCly6H20, ZnS0c7Hz0, CuSO4. 5Hz0, MnSOcHzOand Na2Mo04-2Hz0,O.OOleach; yeast extract, 1.8; and NaOH, approximately 0.18, sufficient to adjust the pH of the solution to 7.0. The lactose was obtained from Sigma Chemical Co. (St. Louis, MO); inorganic salts were from Mallinckrodt (Paris, KY) or Sigma Chemical Co.; yeast extract and Bacto-agar were from Difco Laboratories (Detroit, MI). Flasks were incubated for 48 h at 26 "C and agitated on a rotary shaker at 175 rpm. Serial dilutions (10-107-fold) of each flask were prepared and streaked across isolation agar plates, consisting of LSIM, 18 g/L agar, and 0.01 g/L a-amphotericin as a fungal inhibitor (Fungizone, Gibco Laboratories, Grand Island, NY). A 106 dilution gave the best results; 20 plates were streaked for each sample at this dilution. The plates were incubated for 3-7 days at 26 "C and assayed for exopolysaccharide (gum) on the basis of the following criteria. Various visual, tactile, and chemical tests were employed to identify colonies which appeared to be producing exopolysaccharide. First, colonies were visually screened to determine whether they were opaque, if they exhibited raised, convex, pulvinate, or umbonate morphology, and if they appeared to be mucoid, i.e., slimy or gummy. Second, colonies were screened by tactile evaluation with a stick to determine whether the colonies were viscous, elastic, and gummy. Third, colonies were screened by exposure to two dyes, Cellufluor (Polysciences, Warrington, PA) and Alcian Blue (Sigma Chemical Co.) which bind to some polysaccharides. The entire Cellufluor procedure was done in a dark room. The procedure involved pipetting 2 mL of a 20-50 pg/mL aqueous solution of Cellufluor dye onto agar plates containing small colonies (1-2 mm in diameter). The excess dye was drained off the plates after a 5-min exposure. Colonies which contain @-linkedpolysaccharides are known to appear bright blue upon exposure to long-wave ultraviolet light (345-365 nm) after being stained with Cellufluor (Easson, 1987). The Alcian Blue procedure involved immersing a glass slide covered with dried and heat-fixed cells in an aqueous 0.5% (w/v) Alcian Blue dye solution for 1 min. The dye was then rinsed off, and the slide was dried. A blue appearance surrounding the cells indicates the possible presence of polysaccharide (Norberg and Enfors, 1982). Colonies which passed all of the screening criteria (or most of the screening criteria with at least one strong positive result) were isolated, cultured, and stored at -70 "C for future evaluation. Storage cultures were prepared by adding 10% (v/v) glycerol and 5% (v/v) dimethyl sulfoxide (DMSO) to a cell preparation containing midexponential-phase cells cultured in LSIM and then cooling the mixture to -70 "C at a rate of approximately 1OC/min. These colonieswere subsequently cultured in shake flasks, as described below, to investigate the production of polysaccharide gum. Gum production was estimated by viscosity and cell growth determined by optical density at 660 nm, as described in the following paper (Flatt et al., 1992). Strain Identification. The most promising microorganism (ATCC 55046) was analyzed according to the procedures outlined in Bergey's Manual (Krieg, 1984).The presence of catalase and the production of 3-ketolactose from lactose were determined by the methods described
Biotechnol. Prog., 1992, Vol. 8, No. 4
by Smibert and Krieg (1981). Fatty acid analysis was done by Five Star Laboratories (Branford, CT). Culture Maintenance. A pure culture of ATCC 55046 was prepared and maintained at -70 "C in a solution containing 95 % (v/v) lactose semidefined medium (LSM) and 5 % DMSO. The LSM was identical to the isolation medium (LSIM) except that lactose monohydrate was increased to 45 g/L and (NH4)2S04 was increased to 1.46 g/L. Fermentation Procedures. A single colony of the organism was transferred to a corner-baffled, 1000-mL shake flask containing 300 mL of LSM (see Culture Maintenance), and the flask was incubated at 26 "C on a rotary shaker at 175rpm until the culture was in the midexponential growth phase. A total of 200 mL of medium was aseptically withdrawn from the flask and transferred to a 5.0-L (total volume) mechanically-agitated BioFlo I11 laboratory fermentor (New Brunswick Scientific, New Brunswick, NJ) containing 4.0 L of LSM. Foam was controlled by on-line addition of approximately 0.06 g/L of Mazu DF 60P antifoam (Mazer Chemicals, Gurnee, IL). Two Rushton-typeturbine agitators (both with an impeller diameter to tank diameter ratio of 0.46) were fitted to the agitator shaft to provide mixing and gas transfer to the culture. The fermentation culture was controlled at 26 "C by an external cooling water jacket and a pH of 6.0 by the addition of 4.0 N sodium hydroxide. Sterile air was supplied at 1 volume per volume per minute, and the agitation rate was held at 800 rpm to maintain the dissolved oxygen concentration at greater than 20 % saturation. Oxygen limitation occurred during the latter stage of the fermentation, however, due to the high broth viscosity and incomplete bulk mixing. Polysaccharide Gum Purification. Polysaccharide gum was purified from fermentation broths to study aqueous solution properties and gum composition. The gum was precipitated from the broth by addition of 2propanol to achieve a final 2-propanol concentration of 75% (v/v) and then equilibrated at 4 "C for 24 h. The precipitate was recovered by centrifugation at l8000g for 10min. The precipitate was redissolved in deionized water to achieve a final gum concentration of 2-3 g/L. Cells were removed by centrifugation at 45000g for 1h followed by decantation of the supernatant. The gum was then reprecipitated by addition of 2-propanol to achieve a final 2-propanol concentration of 90% (viv). A higher 2-propanol concentration was required in this step due to the lower ionic strength of the solution. The polysaccharide gum was then dissolved in deionized water to achieve a final gum concentration of 1-2 g/L. Any remaining low molecular weight solutes and salts were removed by one of two methods. For small volumes, the gum solutions were dialyzed against 10 volumes of deionized water for 48 h using rinsed Spectra/Por 2 membrane dialysis tubing, having a molecular weight cutoff of 12 00014 000 (Spectrum Medical Industries, Los Angeles, CAI. The dialysis water was changed daily. For large volumes, the gum solutions were ultrafiltered at a constant concentration which was maintained by the cumulative addition of 5 volumes of deionized water. The solutions were then concentrated to a final concentration of 5-8 g/L by ultrafiltration. A Millipore Pellicon tangential-flow plate system fitted with a 0.5-ft2 100 000 MW cutoff PTHK, polysulfone-type filter (Millipore Corporation, Bedford,MA) was used in the ultrafiltration step. Finally, the deionized material was lyophilized and ground to a coarse white powder, giving a purified preparation of primarily the sodium salt of the polysaccharide gum.
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Compositional Analysis of the Polysaccharide Gum. A method adapted from the general procedure of Kennedy and Sutherland (1987) was used to determine the carbohydrate and organic acid composition of the gum. The overall strategy was to completely hydrolyze the gum into its monomer residue components, while minimizing the amount of hydrolysisbyproducts. The neutral, anionic, and cationic sugars and the organic acids were identified separately using HPLC, proton NMR, and enzymatic procedures. Lyophilized, purified gum was dissolved in deionized water to form a 1%(w/w) solution. A total of 0.75 g of gum solution was placed in a soft glass ampule. A total of 0.75 mL of freshly prepared 2.0 M trifluoroacetic acid was added to the ampule, followed by vigorous agitation to completely disperse the gum. Nitrogen was introduced into the ampule just prior to sealing of the ampule to help maintain a reducing atmosphere during the hydrolysis. Ampules were incubated at 121"C for 60 min. The samples were allowed to cool for 10 min prior to drying of the hydrolysis products at 40-45 "C under a stream of nitrogen gas. The hydrolysis products were then redissolved in water for HPLC or enzymatic analysis or in deuterium oxide for NMR analysis. HPLC Analysis. The neutral sugars of the polysaccharide gum were quantified by HPLC analysis using a Waters Model 600 HPLC (Waters Chromatography, Milford, MA) fitted with a cation-exchange column in the calcium (Sugar-PAK 11,Waters Chromatography) or lead form (Aminex HPX-87P; Bio-Rad Laboratories, Richmond, CA). The separation conditions for the calcium column included a0.5 mL/min flow rate of deionized water eluent, a 10-pL sample injection volume, a column temperature of 90 "C, and a refractive index detector temperature of 35 "C. The separation conditions for the lead column were identical except for a column temperature of 85 "C. The presence of anionic sugars and organic acids in the gum was investigated similarly by HPLC analysis utilizing a cation-exchange column in the H+ form (Aminex HPX87H, Bio-Rad Laboratories). The separation conditions included a 0.5 mL/min flow rate of 0.01 N HzS04 eluent, a lo-pL sample injection volume, a column temperature of 40 "C, and a refractive index detector temperature of 35 "C. Quantification of all components was based upon interpolation of the concentration from the peak area on the basis of a standard curve obtained with authentic standards. Enzymatic Assay. The pyruvate content of the gum was quantified by enzymatic analysis (Kit No. 726-UV from Sigma Chemical Co.), involving an NAD+-linked conversion of pyruvate to lactate by lactate dehydrogenase. The NAD+ formed during the reaction was quantified by measurement of the absorbance at 340 nm. Proton NMR Analysis. The composition of the polysaccharide gum was confirmed by 500-MHz proton NMR analysis (Bruker Instruments). Samples were dissolved in deuterium oxide (Sigma Chemical Co). Hydrogen deuterium oxide (HDO), with a shift of 4.63 ppm, was utilized as the reference for assignment of peak shifts. Proton identification, leading to component identification, was based upon comparison of the sample spectra with spectra of authentic standards. Molecular Weight Estimation by GPC. The weight average molecular weight, M,, number average molecular weight, M,, and polydispersity (M,/M,,) of the polysaccharide gum were estimated by GPC using a Waters 600 HPLC fitted with Ultrahydrogel 2000 and 500 columns
929
(Waters Chromatography) in series. The conditions for the separation included a 0.4 mL/min flow rate of deionized water or 1.0 M KCl eluent, a sample injection volume of 80 pL, a column temperature of 40 "C, and a refractive index detector temperature of 35 "C. Molecular weight information was obtained by comparison of the observed retention time distribution with similar distributions for high molecular weight dextran standards (American Polymer Standards, Mentor, OH) and poly(ethy1eneoxide) (PEO) standards (Waters). The dextran standardsranged in size from 7.5 X lo5to 4.95 X lo6 Da, whereas the PEO standards ranged in size from 2.1 X lo4 to 8.6 X lo5 Da, The polysaccharide molecular weights, estimated from PEO and dextran standard curves, were reasonably similar, despite the need to extrapolate thevalues. We were unable to obtain monodispersed, water-soluble molecular weight standards higher than 5 X 106. The M, and M , of the polysaccharide gum were calculated from GPC results using the Maxima 820 gel permeation chromatography software package (Waters). Rheology Measurements. The steady shear viscosity behavior of aqueous solutions of the g u m s was studied using a Wells-Brookfield RV cone and plate viscometer (Brookfield Engineering Laboratories, Stoughton, MA) fitted with a water bath and Zenith 2-159 personal computer (Zenith Data Systems, St. Joseph, MI) for experiment control and data acquisition. Aqueous solutions of polysaccharide gum were prepared by slowly dissolving a predetermined amount of polysacchride in deionized water under vigorous agitation with a mechanical laboratory stirrer. Solutions were agitated until a homogeneous mixture was obtained, usually for a period of 1-2 h. The pH of each solution was adjusted to 7.0 with the addition of small amounts of 4.0 N NaOH. A 1% (w/w) solution with a pH of 7.0 was the stock solution from which all other solutions were prepared. Acidic or basic solutions were prepared by the addition of small quantities of 4.0 N HC1 or 4.0 N NaOH until the desired pH was reached. Polysaccharide-salt solutions of 0.5 % (w/w) polysaccharide gum were prepared by blending a known amount of the stock gum solution with the required amount of 2.0 M salt solution. The salt solutions were prepared by dissolving a sufficient quantity of either potassium chloride (Fisher Scientific, Fair Lawn, NJ), magnesium chloride, or calcium chloride (Sigma Chemical Co.) in deionized water. The polysaccharideaalt solutions were allowed to equilibrate at 4 "C for 24 h prior to viscosity measurement. The steady shear viscosity profile of each polysaccharide gum solution was determined by utilizing a shearrate cycle in which the shear rate was steadily increased at discrete intervals from a minimum value to a maximum value and then decreased a t discrete intervals back to the minimum value. The rotational speed of the cone was held constant for 30 s a t each step until a constant shear stress value was obtained. The measurements were made at 25 "C using cones having an angle of 3 O and diameters of 2.4 cm (CP-41) and 1.2 cm (CP-52). Shear rates from 0.2 to 180 s-l were obtained under these conditions. The viscometer was calibrated with Newtonian oil standards (Brookfield Engineering Laboratories) either daily or at each change of the cone.
Results and Discussion Identifying Characteristics of the Bacterium ATCC 55046. The most promising lactose-utilizing, gumproducing bacterium was isolated from a soil sample near Mineral Point, WI, which had been regularly treated with
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Table I. Comparison of ATCC 55046 Characteristics with Other Species/Genusesa characteristics ATCC 55046 Klebsiella Erwinia Hafnia alvei Serratia plymuthica +I+ +/ + +/ + aerobic/anaerobicb +/+ +I + + + + + + Gram-negative + + + + motile + + + + + rod + + + + + soil + + + + LPS gum production + + H2S production [+I + + + d growth at 37 "C [+I + + + + + catalase activity +urease activity [-I pectate degradation [+I + produces reddish pigment growth on + + + lactose [+ 1 + + + + d glucose + + + + fructose + + + + galactose + + + + mannose + + + + sucrose + + + +maltose [-I + adonitol arabitol + + Tween 40 + + Tween 80 + + + + L-arabinose + + + cellobiose [-1 [-I + + + + D-mannitol + + + D-melibiose + d methyl 0-glucoside + D-raffinose [+I + +L-rhamnose [+I + + d D-sorbitol + + + ++ D-trehalose xylitol + d malonic acid + d propionic acid + quinic acid + + + succinic acid + + d glycerol [+I -C
+ +
+
a
Serratia liguefaciens +I+
+ + + + + + + -
+
d d
+ + + + +
-
+ + + + [+I
-
+ [-I [+I
+
-
-
d
+ +
The comparison is based on data given in Bergey's Manual of Systematic Bacteriology. Symbols: +, 9O-l00% strains are positive; [+I, [-I, ll-25%; -, &lo%. In the absence of nitrate. Some strains have lactose plasmid.
7 6 4 9 % ; d, 26-75%;
whey and whey permeate. Identification of the bacterium was carried out to determine whether the organism was indeed novel and whether any obvious safety issues might exist. The bacterium was initially erroneously assigned to the genus Rhizobium and deposited with the American Type Culture Collection as Rhizobium sp. ATCC 55046. To obtain a more definitive identification of the organism, the procedures outlined in Bergey's Manual (Krieg, 1984) and fatty acid analysis were carried out. The organism is able to grow and produce polysaccharide gum under anaerobic conditions in the absence of nitrate, as evidenced by the visual appearance of colonies grown on LSM agar plates in an anaerobic jar. Distinct colonies and gum formation could be detected less than 24 h after inoculation, indicating relatively fast growth under anaerobic conditions. This finding, coupled with the fact that the organism produces moderate quantities of acetate, lactate, succinate, and 2,3-butanediol (Flatt et al., 1992))indicates that the organism is most likely a new exopolysaccharide-producing bacterium within the Enterobacteriaceae family (Moat and Foster, 1988). The results of tests to determine to which genus the organism belongs are discussed below. Fatty acid analysis (Table 11)suggests that the organism is closely related to four Enterobacteriaceae genera, with the closest match being Hafnia, followed by Serratia, Yersinia, and Erwinia. The organism is probably not a Yersinia species, as the Yersinia, unlike ATCC 55046, do not produce either polysaccharides or H2S (Brenner, 1984). The phenotypic and biochemical characteristics of the
Table 11. Comparison of Different Genuses with ATCC 55046 from Fatty Acid Profiles using the Euclidian Distancea
genus Erwinia Yersinia
Euclidian Distance llfl 8fl
genus Serratia Hafnia
Euclidian Distance 2fl If1
Euclidian Distance is a measurement of the standard deviation of the fit between the unknown species and a known species from the library. In general, a Euclidian distance of less than 10 would be an indication that the samples are from same species, and less than 25 would indicate that they are from the same genus.
polysaccharide-producing genera of the Enterobacteria (which include the remaining genera suggested by the fatty acid analysis) are compared with those of ATCC 55046 in Table I. The closest match is with the Erwinia. The Klebsiella differ from ATCC 55046 in that they are not motile and do not produce H2S. The Hafnia produce only lipopolysaccharides, whereas ATCC 55046 produces an exopolysaccharide gum containing no lipids. The Serratia are characterized by the production of insoluble orange, pink, or magenta pigments (Brenner, 1984) which are not observed in ATCC 55046. Furthermore, the polysaccharide gum produced by ATCC 55046 contains a greater fraction of mannose and galacturonic acid than the polysaccharides produced by the Serratia and does not contain the heptose, L-fucose,and L-rhamnose present in Serratia polysaccharides (Brenner, 1984). The composition of the polysaccharide gum produced by ATCC 55046, discussed in the next section, is different
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I
galacturonic acid rr,
0.32
galactose P
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0.24
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from the compositions of the polysaccharides produced by other known bacteria (Flatt, 1990; Eden-Green and Knee, 1974; Huang, 1979, 1980; Kang et al., 1977). The presence of a significant amount of mannose and galacturonic acid in the gum distinguishes it from reported Erwinia polysaccharides. In light of the fatty acid analysis and phenotypic and biochemical characteristics of the organism, we conclude that ATCC 55046 is most likely a previously unidentified species of Erwinia. The organism has therefore been redesignated Erwinia spp. ATCC 55046. Chemical and Physical Characterization of the Polysaccharide Gum. Polysaccharide Gum Composition and Molecular Weight. The polysaccharide gum produced by ATCC 55046 on LSM has a molar composition of 47-50% mannose, 27-3096 galactose, 18-23% galacturonic acid, and 0-4 % glucose, on the basis of HPLC and enzymatic analysis of the trifluoroacetic acid (TFA) hydrolysis products from four samples of the purified gum. The gum has a most probable composition of mannose, galactose, and galacturonic acid in the approximate molar ratio of 5:3:2. We believe that the glucose,which is present in trace amounts, is an impurity and is not part of the gum. No modifying organic acid substituents, including acetate, pyruvate, succinate, and glycerate, were detected in any of the analyses. The HPLC chromatogram of the gum hydrolysis products on an H+ cation exchange column is shown in Figure 1. The two major peaks correspond to galacturonic acid and a mixture of galactose plus mannose. The remainder of the peaks have been tentatively identified as small amounts of partially hydrolyzed gum and byproducts of the hydrolysis reaction which appear to result primarily from the degradation of galacturonic acid. A lead cationexchange column was used to resolve the galactose and mannose and to determine the ratio of these two sugars (Figure 2). The tentative composition of the polysaccharide gum was confirmed by 500-MHz proton NMR analysis of the TFA hydrolysis products. The spectrum shown in Figure 3 confirms the presence of galactose, mannose, and galacturonic acid. There are no peaks corresponding to either methyl or methylene protons from organic acid modifying groups in the range of 1.2-2.8 ppm. The only major peak in this region, located at 2.32 ppm, was identified to be a product from the acid-catalyzed degradation of galacturonic acid and corresponds to the peak having a retention time of 15.8min in Figure 1. Estimation of the composition of the polysaccharide on the basis of the relative combined peak heights of the signals corresponding to the a- and 8-anomers of each sugar gives a molar composition of the gum of 42% mannose, 35% galactose, and 23% galactu-
'
'
'
'
6
' . '
.
' '
20 30 40 Retention Time, min
10
Retention Time, min
Figure 1. HPLC chromatogram of the trifluoroacetic acid (TFA) hydrolysis products of the galactomannan polysaccharide gum on an H+ cation-exchangecolumn. The unlabeled peak is thought to be partially hydrolyzed gum and/or byproducts of the hydrolysis reaction.
' . '
1
'
'
,I 50
Figure 2. HPLC chromatogram of the trifluoroaceticacid (TFA) hydrolysis products of the galactomannan polysaccharide gum on a lead cation-exchange column.
ronic acid. These values are somewhat different from those obtained via the HPLC analysis described above but are not as reliable due to the approximate nature of the NMR analysis (peak heights were used since peak areas were not available). The composition of this gum is unique from the reported compositions of other microbial polysaccharide gums (Flatt, 1990). Therefore, we have given this gum the name lactan gum. The composition of lactan gum is related to the compositions of locust bean and guar gums; however, the lactan gum is anionic, whereas locust bean and guar gums are neutral. It is possible that the new gum could function in applications where galactomannans are currently used, such as stabilizing agents for foods and paper wet-end additives and sizing agents. The lactan gum has an estimated M , of 7.0 X 106,an estimated M , of 6.4 X lo6, and an estimated polydispersity of 1.1,on the basis of results of gel permeation (GPC) chromatography using high molecular weight dextran standards. These values are similar to those obtained when the retention times are referenced to poly(ethy1eneoxide) standards. Estimation of the molecular weight from poly(ethylene oxide) standards requires substantial extrapolation and, therefore, was used only to confirm the results using the dextran standards. The M , of the gum is slightly higher than that of a typical xanthan gum sample (Aldrich Chemical Co., Milwaukee, WI), which we estimated by the same method to be 6.0 X 106. This value for xanthan gum is similar to previously reported values (Pace, 1987; Suh et al., 1990). Rheological Properties. The rheological properties of aqueous solutions of the lactan gum were studied to determine potentially useful functions of the gum in commercial applications. Other potential functions of the gum, including film formation and adhesion, were not studied, although qualitative observations of some additional properties are discussed at the end of this section. The steady shear flow behavior of aqueous soldtions of the sodium salt of the gum was determined as a function of gum concentration, added salt type and concentration, pH, and temperature. The steady shear viscosity at 25 "C, pH 7, with no added salt, is shown as a function of shear rate and gum concentration in Figure 4. The flow behavior is pseudoplastic at all of the concentrations studied (0.1-2.0% (w/w)), although the flow behavior becomes more Newtonian at concentrations less than 0.5 % and shear rates below 2 s-l. The flow behavior is best modeled by the power law equation:
+
= T, k(-y)" (1) where k = consistency index (in Pa@), n = flow behavior index (dimensionless), and 70 = yield point (in or Pa.@. T
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1
I
I
5.6
52
1
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I
48
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I
I
I
40
44
I
1 7 6
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72
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1
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1
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20
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1 I0
I'PM
Figure 3. 500-MHz proton NMR spectrum of the trifluoroacetic acid (TFA) hydrolysis products of the galactomannan polysaccharide gum (HDO, hydrogen deuterium oxide reference; man, mannose; galA, galacturonic acid; gal, galactose).
io1
io' io2 Shear Rate, see-1
ioc
io3
Figure 4. Steady shear viscosity vs shear rate at various lactan gum concentrations (25 "C, pH 7, no added salt).
The parameters for this relationship are shown in Figure 5 as a function of gum concentration. The flow behavior index (degree of pseudoplasticity) is higher for lactan gum than for xanthan gum (0.26 vs 0.12 at 1%(w/w) polysaccharide). The flow behavior is thixotropic above 1% (wiw) gum. The gum remains soluble in the presence of common monovalent (sodium and potassium) and divalent (calcium and magnesium) cations present at concentrations of up to 1.00 M. The steady shear viscosity versus shear rate for potassium, magnesium, and calcium salts at 25 "C, pH 7, and 0.5% (w/w) gum are shown in Figures 6, 7, and 8. The viscosity of aqueous solutions of the gum decreases in the presence of various salts, as is typical for most polyelectrolytes. This behavior is believed to be related to contraction of the polysaccharide chain as a result of electrolyte-induced charge shielding, which reduces the repulsion between anionic substituents within the chain. The reduction in viscosity is more noticeable at lower shear rates (lo 9-1). The viscosity appears to reach a minimum somewhere between 0.05 and 0.20 M salt, depending upon the type of salt and shear rate. Upon addition of more salt, the viscosity remains stable or may actually increase slightly.
0
1 'e ',
n
02
os
06
04
I
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02
12
Concentration. wt %
Figure 5. Power law parameters estimated for aqueous solutions of varying concentrations of lactan gum (25 "C, pH 7, no added salt).
I
loll
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10:
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'
" " "
'
" ' " "
lo3 10' lo2 Shear Rate, see-1
'
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Figure 6. Steady shear viscosity vs KCl concentration at 0.5% (w/w) lactan gum (25 "C, pH 7).
The viscosity of aqueous lactan gum solutions remains fairly constant over a pH range of 2-11, as is shown in Figure 9 for a 1.0% gum solution at 25 "C, with no added salt. The viscosity is essentially insensitive to pH from pH 5 to 11but more sensitive to pH below pH 3. The mild viscosity decrease in this region may be due to protona-
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90C.30min.
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0.01M [CaCIZl 0.05M [CaCIZl
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" " " "
s
Figure 10. Temperature stability of aqueous 1.0% (w/w) lactan gum solutions as a function of solution environment (25 OC, 100 8-1 shear rate; all solutions at pH 7, with no added salt, except as noted).
- OM[CaC121
0
A
6
to that of gum arabic which requires protein to function effectively as an adhesive (Glicksman and Sand, 1973).In particular, the "tack" of the gum solutions appears to reach a maximum at a critical ratio of gum to cellular material in the range from 5:l to 3:l (w/w). Solutions of the gum did not form an obvious gel under any of the conditions tested. Solutions at pH 2-11 and containing up to 2.0% (w/w) gum and up to 1.00M salt (potassium, calcium, or magnesium cation) did not form gels in the temperature range from 25 "C to 121 "C. Finally, the gum is readily soluble in cold or hot water. Solubilization rates in aqueous solutions were not quantified due to our inability to produce dried gum material with uniform particle size.
'''.."A
Conclusions
lo2 2
4
6
8
1
0
1
2
PH Figure 9. Steady shear viscosity vs pH a t 1.0% (w/w) lactan gum (25 OC, no added salt).
tion of the carboxylic acid moiety of the galacturonic residues, whose pKa is approximately 3.5-4.5 (McDowell, 1966). To test the thermostability of the lactan gum, gum solutions were exposed to temperatures of 90 "C and 121 "C for varying lengths of time and then cooled to 25 "C. The viscosity at 25 "C after treatment was compared with the viscosity at 25 "C before treatment (Figure 10). Aqueous 1% (w/w) gum solutions, at various levels of pH and ionic strength, exhibited good viscosity stability at 90-121 "C. Gum solutions a t pH 5.0-11, or which contained high levels of potassium or calcium cations, maintained viscosity upon exposure to a temperature of 90 OC for 30 min. The viscosity of solutions at pH 5-11 remained stable after exposure to 121 "C for 15 min; however, the viscosity decreased under these conditions in the presence of 1.0 M salt. The thermostability of the gum was particularly good in alkaline environments. Miscellaneous Properties. Qualitative observations indicate that lactan gum has good adhesive properties, especially in the presence of cellular material. The adhesive nature of this galactomannan gum may be similar
We have isolated a microorganism which is capable of producing a potentially useful polysaccharide gum from lactose. The microorganism, tentatively classified as Erwinia sp. ATCC 55046,is distinct from previously characterized polysaccharide-producingbacteria on the basis of microbiological tests and the unique composition of its exopolysaccharide gum. The anionic galactomannan gum (lactan gum) exhibits several potentially useful properties. The gum has shearthinning behavior and can be modeled by the power law equation. The gum is a more efficient thickener at low concentrations than xanthan gum (less than 0.20%) but a less efficient thickener in a low ionic strength environment at high concentrations. The viscosity is stable between pH 2 and 11, and the gum has good thermostability in the absence of salts. The gum exhibits shearthinning and elastic flow behavior. The combination of moderately low thickening efficiency relative to the high molecular weight of the gum, the inability to form gels, the rapid solubilization rate in aqueous solution, and the lack of organic substituent groups all indicate that the gum is very likely branched (Whistler, 1973). Most microbial polysaccharide gums contain some organic acid substituents, are branched, or are a combination of both, to reduce the crystallinity of the gum (Atkina, 1990). On the basis of the above properties, potential applications of lactan gum include food and nonfood products requiring a moderate degree of thickening, wet-end additives and coating agents for paper products, and binders for building materials and ceramics. The gum may be well-suited for application as a resoiling inhibitor for detergent formulations since the requirements for superior resoiling inhibition agents include anionic char-
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acter, good alkaline and temperature stability, and relatively low thickening efficiency. The useful properties of lactan gum, coupled with the rapid production rate of this gum from lactose relative to other carbohydrate sources, as discussed in the following paper (Flatt et al., 1992), indicate that its production could lead to a partial solution to the problem of whey under utilization.
Acknowledgment We acknowledge Mr. Rich Edwards of the Department of Natural Resources, State of Wisconsin, for assistance in acquiring soil samples. We also acknowledge the input and support of Prof. Norman Olson, Director, Center for Dairy Research, Madison, WI. This study made use of the National Magnetic Resonance Facility at the UWMadison which is supported in part by NIH Grant RR02301 from the Biomedical Research Technology Program, Division of Research Resources. Equipment in the facility was purchased with funds from the University of Wisconsin, the NSF Biological Instrumentation Program (Grant PCM-8415048), the NIH Biomedical Research Technology Program (Grant RR02301), the NIH Shared Instrumentation Program (Grant RR02781), and the U.S. Department of Agriculture. This study was funded by the Wisconsin Milk Marketing Board and the State of Wisconsin Economic Development Fund. Smallscale fermentations were conducted in a BioFlo I11 fermentor which was provided by New Brunswick Scientific, New Brunswick, NJ, as a gift to the University of Wisconsin Bioprocess and Metabolic Engineering Consortium. Literature Cited Atkins, E. Structure of Microbial Polysaccharides Using X-Ray Diffraction. In Novel Biodegradable Microbial Polymers: Proceedings of the N A T O Advanced Research Workshop on N e w Biosynthetic, Biodegradable Polymers o f Industrial Interest from Microorganisms; Dawes, E. A., Ed.; Kluwer Academic Publishers: Dordrecht, 1990; pp 371-386. Brenner, D. J. Facultatively Anaerobic Gram-Negative Rods, Family 1. Enterobacteriaceae. In Bergey’s Manual of S y s tematic Bacteriology; Krieg, N. R., Holt, J. G., Eds.; Williams and Wilkins: Baltimore, 1984; Vol. 1, pp 408-420. Clark, W. S. Status of Whey and Whey Products in The U.S.A. Today. In Trends i n W h e y Utilization,Bull. Znt. Dairy Fed. 1987,212, 6-11. Coton, G. The Utilization of Permeates from the Ultrafiltration of Whey and Skim Milk. Bull. I n t . Dairy Fed. 1980,126,2333. Easson, D. D. A Recombinant DNA Approach to the Design and Synthesis of Novel Polysaccharides. Ph.D. Dissertation, Massachusetts Institute of Technology, Cambridge, MA, 1987; pp 101-102. Eden-Green, S. J.; Knee, M. Bacterial Polysaccharide and Sorbitol in Fireblight Exudate. J.Gen. Microbiol. 1974,81,50912.
Flatt, J. H. Microbial Production of Novel Polysaccharides from Lactose in Whey Permeate. Ph.D. Dissertation, University of Wisconsin, Madison, WI, 1990. Flatt, J. H.; Cooper, T. A.; Gonzalez, J. M.; Dogger, D. E.; Lightfoot, E. N.; Cameron, D. C. An Anionic Galactomannan Polysaccharide Gum from a Newly-Isolated Lactose-Utilizing Microorganism. 11. Fermentation Kinetics and Lactose Transport. Biotechnol. Prog. 1992, following paper in this issue. Glicksman, M.; Sand, R. E. Gum Arabic. In Industrial Gums, 2nd ed.; Whistler, R. L., Ed.; Academic Press: New York, 1973; pp 197-263. Hoogstraten, J. J. The Marketing of Whey Products: A View from Europe. In Trends i n W h e y Utilization,Bull. I n t . Dairy Fed. 1987,212, 17-20. Huang, J.4. A Galactosyltransferase from Erwinia amylovora and Its Possible Role in Biosynthesis of Polysaccharide in Infected Tissues. Physiol. Plant Pathol. 1979, 15, 193-200. Huang, J.-S.Galactosyltransferase Activity in Erwinia stewartii and Its Role in Biosynthesis of Extracellular Polysaccharide. Physiol. Plant Pathol. 1980, 17, 73-80. Kang, K. S.; Veeder, G. T.; Richey, D. D. Zanflo-A Novel Bacterial Heteropolysaccharide. In Extracellular Microbial Polysaccharides; Sandford, P. A., Laskin, A,, Eds.; ACS Symposium Series 45; American Chemical Society: Washington, DC, 1977; pp 211-219. Kennedy, A. F. D.; Sutherland, I. W. Analysis of Bacterial Exopolysaccharides. Biotechnol. Appl. Biochem. 1987, 9, 1217. Krieg, N. R. Identification of Bacteria. In Bergey’s Manual of Systematic Bacteriology; Krieg, N. R., Holt, J. G., Eds.; Williams and Wilkins: Baltimore, 1984; Vol. 1, pp 24-26. McDowell, R. H. J. Alginates and Their Derivatives. SCIMonogr. 1966, 24, 19-32. Moat, A. G.; Foster, J. W. Microbial Physiology, 2nd ed.; J. Wiley & Sons: New York, 1988; pp 190-193. Morr, V. V. Production and Use of Milk Proteins in Food. Food Technol. 1984, 38 (7), 39. Norberg, A. B.; Enfors, S.-0. Production of Extracellular Polysaccharide by Zoogloea ramigera. Appl. Enuiron. Microbiol. 1982, 44, 1231-1237. Pace, G. W. Microbial Gums. In Basic Biotechnology; Bu’lock, J., Kristiansen, B., Eds.; Academic Press: London, 1987; pp 449-462. Smibert, R. M.; Krieg, N. R. In Manual of Methods f o r General Bacteriology; Gerhardt, P., Ed.; American Society for Microbiology: Washington, DC, 1981; pp 417-422. Suh, I. S.; Herbst, H.; Schumpe, A,; Deckwer, W. D. The Molecular Weight of Xanthan Polysaccharide Produced Under Oxygen Limitation. Biotechnol. L e t t . 1990, 12 (3), 201-206. Wells, J. Extracellular Microbial Polysaccharides-A Critical Overview. In Extracellular Microbial Polysaccharides; Sandford, P. A., Laskin, A., Eds.; ACS Symposium Series 45; American Chemical Society, Washington, DC, 1977; pp 299313. Whistler, R. L. Factors Influencing Gum Costs and Applications. In Industrial G u m s , 2nd ed.; Whistler, R. L., Ed.; Academic Press: New York, 1973; pp 5-18. Accepted May 29, 1992.
