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Optimization of Exopolysaccharide Production by Lactobacillus delbrueckii subsp.

bulgaricus RR Grown in a Semidefined Medium


Stacy A. Kimmel, Robert F. Roberts and Gregory R. Ziegler Appl. Environ. Microbiol. 1998, 64(2):659.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1998, p. 659664 0099-2240/98/$04.00 0 Copyright 1998, American Society for Microbiology

Vol. 64, No. 2

Optimization of Exopolysaccharide Production by Lactobacillus delbrueckii subsp. bulgaricus RR Grown in a Semidened Medium
STACY A. KIMMEL, ROBERT F. ROBERTS,*
AND

GREGORY R. ZIEGLER

Department of Food Science, The Pennsylvania State University, University Park, Pennsylvania 16802
Received 11 August 1997/Accepted 25 November 1997

The optimal fermentation temperature, pH, and Bacto-casitone (Difco Laboratories, Detroit, Mich.) concentration for production of exopolysaccharide by Lactobacillus delbrueckii subsp. bulgaricus RR in a semidened medium were determined by using response surface methods. The design consisted of 20 experiments, 15 unique combinations, and ve replications. All fermentations were conducted in a fermentor with a 2.5-liter working volume and were terminated when 90% of the glucose in the medium had been consumed. The population of L. delbrueckii subsp. bulgaricus RR and exopolysaccharide content were measured at the end of each fermentation. The optimum temperature, pH, and Bacto-casitone concentration for exopolysaccharide production were 38C, 5, and 30 g/liter, respectively, with a predicted yield of 295 mg of exopolysaccharide/liter. The actual yield under these conditions was 354 mg of exopolysaccharide/liter, which was within the 95% condence interval (217 to 374 mg of exopolysaccharide/liter). An additional experiment conducted under optimum conditions showed that exopolysaccharide production was growth associated, with a specic production at the endpoint of 101.4 mg/g of dry cells. Finally, to obtain material for further characterization, a 100-liter fermentation was conducted under optimum conditions. Twenty-nine grams of exopolysaccharide was isolated from centrifuged, ultraltered fermentation broth by ethanol precipitation. Interest in exopolysaccharide (EPS)-producing lactic acid bacteria has increased recently because these food grade organisms produce polymers important in determining the rheological properties of dairy products and may have application in nondairy foods. When added to food products, polysaccharides function as thickeners, stabilizers, emulsiers, gelling agents, and water binding agents (24). Evaluation of the functional attributes of potentially useful EPSs in foods requires that the material be available in sufcient quantities. This generally requires scale-up from bench to pilot scale fermentations once optimal conditions have been identied. Optimization of the growth environment is important to achieving maximal EPS production by organisms such as Xanthomonas, Pseudomonas, and Rhizobium spp. (2, 19, 21, 23). Published efforts to optimize EPS production in lactic acid bacteria have included studies evaluating the effects of such environmental conditions as temperature and pH (6, 9, 10, 15, 16, 20, 28). A number of these studies examined the effect of temperature by using various EPS-producing strains of Lactobacillus delbrueckii subsp. bulgaricus. Garcia-Garibay and Marshall (9) found that specic polymer production (equivalent milligrams of dextran per CFU) by strain NCFB 2772 grown in skim milk was greater at a temperature (48C) higher than the optimum for growth (37 to 42C). Greater specic polymer production (milligrams per gram of cells [dry weight]) at a higher temperature (45C) was also found by Grobben et al. (10), who examined EPS production by the same strain in dened medium. Mozzi et al. (16) found a correlation between the optimum growth temperature (37 to 42C) and maximum polymer production by strain CRL 870. In contrast, Schellhaass (20) reported higher polymer production by strain RR at temperatures below the optimum for growth. Van den Berg et al. (28) conducted a study evaluating the effects of temperature and pH on EPS production by L. sake 0-1. They found, by using a one-variable-at-a-time (OVAT) approach, that optimal EPS production occurred at 20C and pH 5.8. Another study by Mozzi et al. (15) found that maximum polymer synthesis (488 mg/liter) by L. casei CRL 87 at 30C occurred at pH 6.0. However, optimum specic production (EPS produced per gram [dry weight] of cells) and EPS yield (grams of EPS 100/grams of sugar consumed) were found at pH 4.0. For some EPS-producing bacteria, such as Xanthomonas, Pseudomonas, and Rhizobium spp., nitrogen-limiting conditions result in increased EPS production (3, 25). The effect of nitrogen concentration on EPS production by lactobacilli has not been examined. L. delbrueckii subsp. bulgaricus RR is a common EPS-producing strain. The effect of using L. delbrueckii subsp. bulgaricus RR as a starter culture on the rheological properties of milk gels (yogurt) has been examined under various conditions (12, 20, 26, 27). In addition, the monosaccharide composition and repeating structure of the EPS produced by L. delbrueckii subsp. bulgaricus RR have been determined (11). However, characterization of the interaction between milk proteins and the EPS produced by L. delbrueckii subsp. bulgaricus RR, as well as evaluation of the functional effects of the EPS in nonfermented food systems, has been hampered by the unavailability of sufcient quantities of the polymer. The objectives of the present work were to determine the optimum temperature, pH, and Bacto-casitone (nitrogen; Difco Laboratories, Detroit, Mich.) concentration for production of EPS by L. delbrueckii subsp. bulgaricus RR in a semidened medium by using a response surface experimental design and to use these conditions to produce EPS for further characterization.
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* Corresponding author. Mailing address: Department of Food Science, The Pennsylvania State University, 111 Borland Laboratory, University Park, PA 16802. Phone: (814) 863-2959. Fax: (814) 8636132. E-mail: RFR3@psu.edu.

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MATERIALS AND METHODS

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with nitrogen during sterilization to obtain an anaerobic environment. Temperature, agitation, and pH were monitored and controlled with a Macintosh computer (Apple Computer Inc., Cupertino, Calif.) and a program developed inhouse by using the Lab View development system (National Instruments, Austin, Tex.). A model 2700 Select Biochemistry Analyzer (YSI Biochemical Products, Yellow Springs, Ohio) was used to monitor glucose utilization throughout fermentation and recovery. At the endpoint (90% of glucose utilized), agitation was increased to 350 rpm and the temperature of the vessel was raised to 100C. After 15 min at 100C, the vessel was cooled to 37C and 100 ml of a 5% (wt/vol) pronase E (EC 3.4.24.31; Sigma Chemical Co.) solution was added. After 60 min at 37C, 250 ml of 80% (wt/vol) trichloroacetic acid was added and the vessel was cooled to 17C. After treatment, fermentation broth was pumped to a Sharples A316Y centrifuge (Sharples Inc., Warminster, Pa.) and centrifuged at 16,800 rpm to remove cells and protein. EPS was concentrated by ultraltration of the claried fermentation broth by using a regenerated cellulose membrane (PLGC; 50-ft2 surface area; molecular weight cutoff, 10,000; Millipore Corp., Bedford, Mass.) in a 50 RL Process Ultraltration System (Millipore Corp.). Low-molecular-weight solutes were removed by dialtration of the EPS-containing retentate with distilled water until the conductivity of the permeate approached that of distilled water (YSI model 34 conductance-resistance meter). The EPS-containing retentate was placed in a 20-liter glass carboy, and the EPS was precipitated by adding 2 volumes of chilled 95% ethanol and then stored for 2 days at 4C. The top layer of liquid was decanted, and the bottom layer, which contained the precipitated EPS, was placed in bottles and centrifuged at 8,000 g and 4C for 20 min. The pellet was removed, resuspended in distilled water, and lyophilized ( 40C). The protein content of the dried material was measured by the Bradford method (1), the total carbohydrate concentration was determined by the phenol-sulfuric acid method (7), and the moisture content was measured by weighing material before and after freeze-drying ( 40C).

Bacterial strain and culture preparation. L. delbrueckii subsp. bulgaricus RR was originally obtained from the laboratory of H. A. Morris (University of Minnesota, St. Paul) and maintained in MRS broth (Difco). Stock cultures were prepared by mixing a pure culture grown for 12 to 16 h at 42C in MRS broth with an equal volume of a 10% glycerol solution and storing the mixture at 75C. Working cultures were prepared by transferring 0.5 ml of the frozen stock culture to 10 ml of MRS broth and incubating it for 12 to 18 h at 40C. The working culture of strain RR was transferred (1% [vol/vol]) to 20 ml of MRS broth and incubated for 12 to 18 h at 40C. This preculture was centrifuged at 8,000 g and 4C for 10 min and washed twice with 20 ml of sterile distilled water. The resulting cell suspension (in sterile distilled water) was used to inoculate larger volumes (2.0 liters) of semidened medium (SDM). Culture medium. The SDM used as a base for EPS experiments had the following composition (in grams per liter): dextrose, 20; Tween 80, 1; ammonium citrate, 2; sodium acetate, 5; MgSO4 7H2O, 0.1; MnSO4, 0.05; K2HPO4, 2; yeast nitrogen base without ammonium and amino acids (Difco), 5; Bacto-casitone (Difco), 10. The pH of all media was adjusted to 6.5 0.2 prior to sterilization by heating for 15 min at 121C. Glucose was sterilized separately and aseptically added to the medium. Previous work in our laboratory indicated that generation times for strain RR in MRS and SDM did not differ signicantly at 42C and that the medium provided minimal interference with the assays used to quantify EPS (data not shown). Experimental design. To determine the optimum conditions for EPS production by strain RR, a response surface experimental design using a quadratic model was created by using ECHIP (ECHIP, Inc., Hockessin, Del.) (29). Three variables were included in the model: the temperature (35 to 45C), pH (4 to 6), and Bacto-casitone (nitrogen) concentration (10 to 30 g/liter) of the growth medium. The design contained 20 experiments, 15 unique combinations, and ve replications (see Table 1). Fermentations. All optimization fermentations were conducted in a 2.5-liter working volume Bio-ow III (New Brunswick, Edison, N.J.) fermentation vessel. After inoculation, the pH declined from 6.5 to the set point for that experiment due to lactic acid production. The pH was then maintained at the set point by addition of 5 N NaOH. Agitation was maintained at 200 rpm throughout the fermentation. Immediately prior to inoculation, the medium was sparged with nitrogen for 20 min at a rate of 1 liter/min. Samples (approximately 30 ml) were removed at regular intervals and analyzed for growth, glucose, and EPS as described below. All fermentations were terminated when 90% of the initial glucose had been consumed. Measurement of growth. Growth was monitored spectrophotometrically (650 nm) and by plating suitable dilutions on MRS agar, followed by anaerobic incubation for 48 h at 42C. For the prole experiment, cell dry weight determinations were conducted on duplicate 10-ml samples of fermentation broth. Each sample was centrifuged (8,000 g for 10 min) and washed twice with distilled water. The pellet was resuspended in 5 ml of distilled water, dried at 97C for 5 h, and subsequently weighed. Glucose analysis. The glucose content of the medium during fermentation was monitored by high-performance liquid chromatography. The chromatographic system consisted of a Waters 510 solvent delivery system (Waters Corp., Milford, Mass.), a Rheodyne 7125 sample injector (20- l loop; Rheodyne Inc., Cotati, Calif.), a Bio-Rad IG Cation H guard column (Bio-Rad, Hercules, Calif.), an Aminex HPX-87H column (300 by 7.8 mm; Bio-Rad), and a Waters 410 differential refractometer (Waters Corp.). The column and detector were maintained at 35C. The solvent, 0.005 M sulfuric acid, was delivered at a ow rate of 0.6 ml/min. Data were collected by using the SSI Vision IV data collection system (Scientic Systems Inc., State College, Pa.). Glucose was quantied by relating the peak area to a standard curve. Quantication of EPS. The procedure used for EPS quantication was based on that described by Gancel and Novel (8). Approximately 10 g of culture medium was accurately weighed into a 50-ml centrifuge tube and then heated in a boiling water bath for 10 min to inactivate enzymes potentially capable of polymer degradation (4, 5). Samples were cooled to room temperature, 100 l of a 5% (wt/vol) pronase E (EC 3.4.24.31; Sigma Chemical Co., St. Louis, Mo.) solution was added, and the mixture was incubated for 1 h at 37C in a water bath shaker. Following protein digestion, 250 l of 80% (wt/vol) trichloroacetic acid was added and samples were mixed well and stored at 4C for a minimum of 30 min and then centrifuged (8,000 g for 20 min) to remove cells and protein. The supernatant was decanted into dialysis tubing (molecular weight cutoff, 6,000 to 8,000) and dialyzed for 48 h against four changes of distilled water. All assays were performed in duplicate, and the same procedure was done with uninoculated medium. The carbohydrate concentration of the retentate was determined by using the phenol-sulfuric acid method (7). The carbohydrate assay was calibrated by using a mixture of D-galactose, D-glucose, and L-rhamnose (5:1:1) (11), and results are reported as milligrams of carbohydrate per liter. The values shown for EPS were calculated by subtracting the amount of background interference in uninoculated medium (approximately 44 mg of carbohydrate/liter) from the amount in fermented broth. Large-scale production and recovery of EPS. Large-scale production of EPS was conducted in a 100-liter (working volume) fermentation vessel (Bio-Service, Allentown, Pa.). Agitation was maintained at 100 rpm, and the vessel was ushed

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RESULTS Optimization of EPS production. A response surface experimental design was used to determine the optimum temperature, pH, and Bacto-casitone concentration for EPS production by strain RR. Results obtained from all of the experiments in the design are presented in Table 1. Run 18 (trial 9) was excluded from the data analysis because growth conditions were too stringent and the endpoint (90% of glucose utilized) was not reached within 168 h. The optimum temperature, pH, and Bacto-casitone concentration for EPS production were determined to be 38C, 5, and 30 g/liter, respectively (Fig. 1), with a predicted optimum production of 295 mg of EPS/liter. The actual yield under these conditions was 354 mg of EPS/ liter, which was within the 95% condence interval predicted (217 to 374 mg of EPS/liter). A plot of the results indicated that higher EPS production might be obtained if the Bactocasitone concentration was greater than 30 g/liter (Fig. 2). To investigate, an experiment was conducted at the optimum temperature (38C) and pH (5.0) with a Bacto-casitone concentration of 40 g/liter. The EPS production under these conditions was found to be 324 g/liter, lower than the amount of EPS produced under optimum conditions (Table 1). When this result was included in the analysis, the predicted optimum and the signicant relationships among growth conditions and response variables did not change. Table 2 provides the coefcients of the statistical models derived from response surface methods, as well as the statistical signicance of each term. To prole the optimum conditions for EPS production by strain RR, an experiment was conducted at 38C, pH 5, and 30 g of Bacto-casitone per liter. Growth and EPS production were monitored throughout the fermentation. The prole showed that EPS production was growth associated, i.e., EPS production followed the growth of the organism, with a specic production at the endpoint of 101.4 mg of EPS/g of dry cells and a specic production rate during growth of 0.472 mg of EPS/g of cells/h (see Fig. 4). Isolation and recovery of EPS. To obtain material for further characterization, a 100-liter fermentation was conducted by using the optimum conditions for EPS production (38C, pH 5, 30 g of Bacto-casitone per liter). Following ultraltration

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TABLE 1. Results of fermentation experiments done to optimize EPS production by L. delbrueckii subsp. bulgaricus RR
Triala Runb Temp (C) pH Bacto-casitone concn (g/liter) Time to endpoint (h)c EPS concn (mg/liter)d ml endpoint

1 1 2 2 3 3 4 4 5 5 6 7 8 9 10 11 12 13 14 15 16e 17f
a b

6 13 9 14 3 19 12 16 2 10 17 11 8 18 5 15 1 20 7 4 21 22

35 35 45 45 35 35 35 35 45 45 40 35 40 35 45 45 40 40 45 35 38 38

6 6 5 5 6 6 4 4 6 6 6 5 5 4 4 6 6 4 5 5 5 5

30 30 10 10 10 10 30 30 30 30 20 20 30 10 20 20 10 10 30 10 30 40

17.50 17.16 17.80 16.00 26.60 25.50 59.16 58.58 9.42 9.16 12.08 21.16 15.08 30.40 9.42 14.91 41.00 11.41 38.25 15.16 14.00

268.00 245.68 203.44 169.23 195.93 146.25 268.84 247.54 154.23 181.45 194.23 245.54 279.60 124.25 139.20 254.31 210.19 178.09 143.34 354.23 324.27

8.20 8.08 7.90 7.98 6.43 6.22 4.35 4.59 8.71 8.96 8.60 7.70 8.27 6.67 8.86 8.33 5.72 8.29 7.01 8.34 8.53

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Each trial with a different number indicates a unique set of experimental conditions. Run signies the order in which the experiments were conducted. c Time at which 90% of the glucose in the growth medium was utilized as detected by high-performance liquid chromatography. d Calculated by subtracting the amount of background interference in uninoculated medium from the amount in fermented broth. e Results of an experiment conducted to verify the predicted optimum conditions. f Results of an experiment conducted with Bacto-casitone at 40 g/liter.

and dialtration to concentrate the EPS and remove low-molecular-weight materials, ca. 5 liters of EPS-containing retentate was obtained. Following ethanol precipitation of the retentate and freeze-drying, 29.2 g of semipure polysaccharide was obtained (Table 3).

DISCUSSION Optimization of EPS production. This study was designed to examine three growth conditions (temperature, pH, and Bacto-casitone concentration) likely to affect EPS production. The inuence of the carbon source on EPS production was not examined because other studies have shown, in general, that glucose (10 to 20 g/liter) provides the highest yield of EPS (6, 14, 17).

FIG. 1. Contour plot of EPS production by L. delbrueckii subsp. bulgaricus RR at 30 g of Bacto-casitone per liter as a function of temperature and pH. Contour lines with numbers are signicantly different (P 0.05). The diagonal line is the design boundary; i.e., experimental conditions below this line were not included in the design.

FIG. 2. Three-dimensional plot of EPS production by L. delbrueckii subsp. bulgaricus RR at pH 5.0 as a function of temperature and Bacto-casitone concentration.

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KIMMEL ET AL. TABLE 2. Coefcients determined for response models


Response (y)a Parameter EPS concn (mg/ml) CFU/ml Time to endpoint (h)b

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Constant ( 0) Temp ( 1) pH ( 2) Casitone ( 3) Temp pH ( 4) Temp Casitone ( 5) pH Casitone ( 6) Temp2 ( 7) pH2 ( 8) Casitone ( 9) r2g Ph

235.82 4.64c NIe 2.34c NI 0.57c NI 2.17f 20.91 0.24 0.838 0.011

1.8 1.8 1.75

108 107c 108d NI 3.4 107f NI 6.3 106 NI 7.5 107 9.4 105 0.923 0.0005

12.8 1.7d 14.5d 0.3c 0.9f NI 0.2 0.2f 9.9d 0.05c 0.983 0.0000

a Y 40) 5) 20) 40) 0 1(T 2(pH 3(casitone 4(T 40)(casitone 20) 5)(casitone 20) (pH 5) 5(T 6(pH 7(T 2 2 2 5) 20) , where T is the temperature of 40) 8(pH 9(casitone fermentation (C), pH is the pH of the medium during fermentation, and casitone is grams of Bacto-casitone per liter of growth medium. b Time at which 90% of the glucose in the growth medium was utilized by the organism. Time to endpoint had a signicant lack of t (P 0.05) with the response surface model. c Signicantly different at P 0.05. d Signicantly different at P 0.001. e NI, not included. Inclusion of this coefcient in the model did not improve the t. f Signicantly different at P 0.01. g Regression coefcient of the model to predict the particular response variable. h P value for the model to predict the particular response variable.

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FIG. 4. Prole of EPS production, cell dry weight, and glucose utilization by L. delbrueckii subsp. bulgaricus RR grown under optimum conditions for EPS production (38C, pH 5.0, 30-g/liter Bacto-casitone). E, EPS; , glucose; , cell dry weight.

The optimum temperature for EPS production was within the optimum growth range for strain RR (37 to 45C) (13). Published work examining the effect of temperature on EPS production by lactic acid bacteria has been contradictory. Some reports found greater amounts of EPS produced at temperatures within the optimum growth range (9, 10), while others have suggested that more EPS is produced at growth temperatures that are less than the optimum (20). Discrepancies in

the literature exist for a variety of reasons, including different ways of measuring EPS, different growth media, conditions and times of measurement, lack of pH control, and various means of expressing EPS production (milligrams of EPS, milligrams of EPS per liter, milligrams of EPS per CFU). Little work has been done examining the effect of pH on EPS production by lactobacilli, and none has been conducted with the organism used in this study. Mozzi et al. (15) measured maximum polysaccharide synthesis (488 mg/liter) and the highest cell numbers at a constant pH of 6.0 for L. casei CRL 87. In addition, the amount of EPS (milligrams per liter) was 3.6 times as high in fermentations with pH control as in those without pH control. In a study conducted with L. sake 0-1, the optimum pH for EPS production was 5.8 but higher cell numbers were achieved at pH 6.2 (28). From this result, the authors concluded that conversion of sugar to EPS is more efcient at pH 5.8 but sugar is more efciently converted to biomass at pH 6.2. For some EPS-producing bacteria, such as Xanthomonas,

TABLE 3. Recovery table for EPS produced by L. delbrueckii subsp. bulgaricus RR


Sampling period and material tested Vol of medium (liters) EPS concn EPS yield (g)a EPS recovery (%)b

During recovery Fermentation broth 97.00 Concentrated fermentation 5.32 brothd After recovery Freeze-dried precipitate
a b

367.84c 6,647.27c

35.68 35.36

100.0 99.1

964.00

22.0e 29.20f

81.84

FIG. 3. Contour plot of productivity (product) with 30 g of Bacto-casitone per liter as a function of temperature and pH. Contour lines with numbers are signicantly different (P 0.05). The diagonal line is the design boundary; i.e., experimental conditions below this line were not included in the design.

Yield (grams) EPS (grams per liter) volume of medium (liters). Recovery (%) grams of EPS/grams of EPS contained in fermentation broth 100. c In milligrams per liter. d After ultraltration and dialtration. e In milligrams per gram. f The EPS had a protein content of 1.42% (0.43 0.11 g) and a moisture content of 0.3% (0.0073 0.0005 g).

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Pseudomonas, and Rhizobium spp., nitrogen limitation results in increased EPS production (3, 25). This was not the case for strain RR. Optimum EPS production occurred in a growth medium containing three times as much Bacto-casitone (30 g/liter) as that needed to support growth (10 g/liter). Increasing the Bacto-casitone content in SDM to 40 g/liter did not result in an increased level of EPS production (Table 1). This may have been because other nutrients became limiting in batch culture. A response surface experimental design employing a quadratic model was used in this study. This type of model is logical because factors that affect microbial growth often show a single maximum. Statistical analysis provided information about the effect of growth conditions on the response variables (Table 2). A signicant relationship was found between temperature (temperature and temperature squared) and EPS production. In general, the relationship between temperature and the growth of microorganisms shows a single maximum but is asymmetric (22), as was the relationship observed here. The relationship between pH and microbial growth often shows a broad maximum (22). In this study, it is likely that pH did not signicantly affect EPS production because the range used in this study (4 to 6) was on the relatively at part of the curve. In fact, Fig. 1 suggests that the optimum temperature and pH conditions for EPS production exist within the ellipse bounded by 36 to 39C and pH 4.5 to 5.5, rather than at a specic point. Finally, there was a signicant relationship between the Bactocasitone concentration and EPS production, which is shown in Fig. 2. A number of growth conditions and their interrelationships were signicantly related to the number of CFU per milliliter and time to endpoint. It is generally known that growth conditions such as those examined in this study have an effect on these variables. Productivity, dened as production of the maximum amount of a product in a minimum amount of time, is an important variable from an industrial perspective. To determine conditions for optimum productivity, a combined response variable, designated productivity, was calculated as the weighted sum of the individual responses EPS concentration and time to endpoint (Table 1). By using the EChip optimization routine (29), individual responses are scaled so they can be fairly combined, and the maximum of the combined response is the simultaneous optimum of the individual responses. Individual responses are subjectively weighted to reect their relative importance. In this case, EPS concentration and time to endpoint were equally weighted. Optimum productivity was found at 39C, pH 5.6, and 30 g of Bacto-casitone per liter (Fig. 3). Under these conditions, the model predicted that 236 mg of EPS/liter would be produced in 12.45 h. In the case of expensive medium components, laborious preparation procedures which result in a high ratio of preparation time to fermentation time, or high residual processing costs, it would be appropriate to weight EPS production higher than time to endpoint, thereby reducing the residual substrate (at the cost of longer fermentation times). Previous reports examining the effect of more than one condition on EPS production have utilized an OVAT approach (28). An OVAT approach assumes no interaction among the variables being investigated. Response surface methodology allows simultaneous evaluation of many growth conditions and also examines interaction among the variables (Table 2). Prole under optimum growth conditions. A prole of the fermentation conducted by using the optimum conditions for EPS production showed that EPS production was growth associated (Fig. 4). Similar results were obtained by Grobben et al. (10) by using strain NCFB 2772. This result suggests that

optimizing conditions for growth of the EPS-producing organism would result in maximal production of EPS. Growth association of EPS production does not occur with some other EPS-producing strains, such as Xanthomonas and Alcaligenes spp. (25). Isolation and of recovery of EPS. A large-scale fermentation conducted under optimum conditions for production yielded 29.2 g of crude EPS. Even on scale-up, the amount of EPS produced by strain RR (367.84 mg of EPS/liter; Table 3) was within the 95% condence interval (216 to 374 mg of EPS/ liter) predicted by the response surface model. This is signicant, since scale-up is often difcult due to changes in surfaceto-volume ratios. In this case, scale-up may have been more straightforward, since oxygen transfer is not the problem under anaerobic conditions that it may be under aerobic conditions. The greatest loss during recovery of crude EPS was probably due to the method of harvesting the EPS, in which only the bottom layer of precipitated material was centrifuged following ethanol precipitation (Table 3). The remaining EPS may have been present as ne particles dispersed throughout the ethanol-retentate mixture. A ltration step to recover the EPS might result in higher recovery rates than the process used in this study. The protein content of crude material was determined to give an estimate of purity. The material was found to contain 1.42% (0.43 0.11 g) protein. It is likely that the protein was carried over from medium ingredients during recovery and is not tightly bound to EPS. Oda et al. (18) also found a small amount of protein contaminating polysaccharide isolated from L. helveticus var. jugurti.
ACKNOWLEDGMENTS This research was supported in part by grants from the Pennsylvania Dairy Promotion Program and the American Dairy Association and Dairy Council Mid East. We thank Mark Signs for his assistance with fermentation scale-up.
REFERENCES 1. Bradford, M. M. 1976. A rapid and sensitive method for the quantication of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248254. 2. Breedveld, M. W., L. P. T. M. Zevenhuizen, H. C. J. Canter Cremers, and A. J. B. Zehnder. 1993. Inuence of growth conditions on production of capsular and extracellular polysaccharides by Rhizobium leguminosarum. Antonie Leeuwenhoek 64:18. 3. Cerning, J. 1990. Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiol. Rev. 87:113130. 4. Cerning, J., C. Bouillanne, and M. J. Desmazeaud. 1988. Extracellular polysaccharide production by Streptococcus thermophilus. Biotechnol. Lett. 10: 255260. 5. Cerning, J., C. Bouillanne, M. Landon, and M. Desmazeaud. 1992. Isolation and characterization of exopolysaccharides from slime-forming mesophilic lactic acid bacteria. J. Dairy Sci. 75:692699. 6. Cerning, J., C. M. G. C. Renard, J. F. Thibault, C. Bouillanne, M. Landon, M. Desmazeaud, and L. Topisirovic. 1994. Carbon source requirements for exopolysaccharide production by Lactobacillus casei CG11 and partial structure analysis of the polymer. Appl. Environ. Microbiol. 60:39143919. 7. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350356. 8. Gancel, F., and G. Novel. 1994. Exopolysaccharide production by Streptococcus salivarius ssp. thermophilus cultures. 1. Conditions of production. J. Dairy Sci. 77:685688. 9. Garcia-Garibay, M., and V. M. E. Marshall. 1991. Polymer production by Lactobacillus delbrueckii ssp. bulgaricus. J. Appl. Bacteriol. 70:325328. 10. Grobben, G. J., J. Sikkema, M. R. Smith, and J. A. M. de Bont. 1995. Production of extracellular polysaccharides by Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 grown in a chemically dened medium. J. Appl. Bacteriol. 79:103107. 11. Gruter, M., B. R. Leeang, J. Kuiper, J. P. Kamerling, and F. G. Vliegenthart. 1993. Structural characterisation of the exopolysaccharide produced by Lactobacillus delbrueckii subspecies bulgaricus rr grown in skimmed milk. Carbohydr. Res. 239:209226.

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