AN ABSTRACT OF THE THESIS OF
Nilo Fatemeh Youssef-Hakimi for the degree of Master of Science in Food
Science and Technology presented on November 17. 1992.
Title: Production and Characterization of Extracellular Polysaccharides Produced
by Lactobacillus plantarum
Abstract approved: / 7 /Dr./ J.t Antonio\ Torres
A Lactobacillus plantarum strain (335) was isolated for its ability to produce an extracellular polysaccharide which increased the viscosity of the growth medium. MRS was the medium chosen for initial characterization studies of microbial growth and polysaccharide production. A non-producing L. plantarum strain (337) was used as control. Under various temperature conditions, the polysaccharide producing strain had the same generation time, was able to ferment the same sugars and lowered the medium pH at the same rate as the non- producing strain. After 4 d incubation, when grown on MRS agar at 250C, the two strains differed in colony appearance. The polysaccharide producing strain (335) formed larger colonies with viscous material on the surface and around the colonies. Electron microscopy of the producing strain showed presence of extracellular material in the medium and also attached to the colonies. An increase in viscosity was observed after 10-20 h incubation at 350C with the polysaccharide producing strain. This was not observed in MRS inoculated with the non-producing strain. Identical observations were made when using a simple defined medium (RPMI-1640) for growth and viscosity change determinations.
HPLC analysis of the lyophilized precipitated polysaccharide suggested the presence of mannose and glucose in the polysaccharide structure. However, similar peaks were present in the chromatogram for the non-polysaccharide producing strain. This observation and the poor chromatographic separation prevented a clear confirmation of the presence of mannose and glucose in the polysaccharide structure. TLC analysis showed no significant differences in the number of spots and in sugar migration rates (Rf values) between the material recovered after growth of the polysaccharide producing and non-producing strain.
An additional 20 L. plantarum strains were inoculated in acid whey to determine their ability to utilize lactose as the sole sugar source. All strains examined grew on this medium, however, after 10-15 h of incubation, only strains
59 and 335 caused an increase in the viscosity of the medium. These two strains, along with the non-polysaccharide producing strain (337), were inoculated in a continuous fermenter to determine lactose utilization under controlled temperature (350C) and pH (6.0). Strain 337 lowered the lactose content in acid whey from 4.6% to 2.6%, while strain 335 and 59 reduced it to 2.2% and 1.5%?, respectively. All strains showed successful growth in acid whey, maintaining 108 to 109 CFU/mL during the course of the controlled fermentation. Production and Characterization of Extracellular Polysaccharides
Produced by Lactobacillus plantarum
by
Nilo Fatemeh Youssef-Hakimi
submitted to
Oregon State University
in partial fulfillment of
the requirement for the
degree of
Master of Science
Completed November 17, 1992
Commencement June 1993 APPROVED:
ssociate Professor, ^ood Science and Technology, in charge of major
7 ■ ~g Head, Department of Food Science and Technology
r(y » ^v\,0 r f * * " ^v y " ^mj Dean of Graiduate Schi
Date thesis is presented: November 17. 1992
Typed by: Reitha McCabe ACKNOWLEDGMENTS
I wish to express my sincere appreciation and gratitude to my major professor, Dr. Antonio Torres for his constant guidance, encouragement and support during my studying in the Department of Food Science and Technology.
Special acknowledgements are made to the members of the graduate committee, Drs. Mark Daeschel, Janine Trempy, Daniel Selivonchick, and Hillary
Carpenter for their advice and critical review of this dissertation.
I would like to give special thanks to all faculty, staff and graduate students of the Department of Food Science and Technology.
The financial support from the Western Dairy Food Center is gratefully acknowledged.
Special recognition is given to my parents, Abdullah and Shahin for their love, understanding and providing me with an excellent educational opportunity.
I would also like to thank my brother Ali for his constant support and encouragement. TABLE OF CONTENT
Page
INTRODUCTION 1
LITERATURE REVIEW 3
Whey as food and beverage ingredient 3
Whey bioconversions 4 Alcohol from whey 5 Single cell protein and biomass 5 Lactic acid production 6 Microbial solvent production 6
Microbial polysaccharides 7 Extracellular polysaccharides 8 Structural analysis of microbial polysaccharides 8 Polysaccharides applications in foods 9
MATERIALS AND METHODS 10
Polysaccharide producing bacteria 10 Culture media and growth of bacteria 11 Electron microscopy characterization 12 Extraction and purification of polysaccharide 12 Polysaccharide hydrolysis 13 Protein determination 13 Chromatography 14 Viscosity measurements 15
Acid whey fermentation by L. plantarum strains 15 Test tube studies 16 Controlled pH fermentation 16
RESULTS AND DISCUSSION 19
Polysaccharide producing bacteria 19 Culture media and growth of bacteria 19 Electron microscopy characterization 20 Polysaccharide production 21 Thin layer chromatography 22
Acid whey fermentation 24
CONCLUSIONS 47
BIBLIOGRAPHY 49 LIST OF FIGURES
Page
Figure 1.1 Procedure for isolation and purification of polysaccharide . . 17
Figure 1.2 Protein determination by the Lowry assay. Calibration curve of optical density versus BSA (mg) 18
Figure II.l Arrhenius plot for the specific growth rate of polysaccharide producing and non-producing strains of L. plantarum in MRS 29
Figure II.2 Colony appearance of polysaccharide producing (1) and non- producing (2) L. plantarum strains after 5d at 250C incubation in MRS agar 30
Figure II.3 Glucose disappearance during growth in MRS of the polysaccharide producing and non-producing L. plantarum strains 31
Figure II.4 pH changes during growth of the polysaccharide producing and non-producing L. plantarum strains in a defined medium (RPMI 1640) supplemented with 2% MRS broth 32
Figure II.5 TEM micrographs of L. plantarum a. polysaccharide producing strain b. non-polysaccharide producing strain 33
Figure II.6 SEM micrographs of L. plantarum a. polysaccharide producing strain b. non-polysaccharide producing strain 34
Figure II.7 Changes in MRS viscosity after inoculation with polysaccharide producing and non-producing strains of L. plantarum 35
Figure II.8 Changes in the viscosity of a defined media (RPMI 1640) enriched with 2% MRS solution during growth of polysaccharide producing and non-producing L. plantarum strains 36 Figure II.9 HPLC chromatographs of the recovered precipitate after fermentation with the polysaccharide producing strain of L. plantarum in modified MRS with glucose (1), lactose (2) and sucrose (3) as the sole sugar source 37
Figure 11.10 HPLC chromatographs of the recovered precipitate after fermentation with the polysaccharide producing strain of L. plantarum in modified MRS with galactose (1), fructose (2) and maltose (2) as the sole sugar source 38
Figure 11.11 Rf values on TLC plates spotted with sugar standards, and hydrolyzed recovered polysaccharides after growth of the producing (P+) and non-producing (P-) L. plantarum strains in MRS and 1640 media. Spotting locations correspond to glucose (1), polysaccharide producing strain in 1640 (2), non- producing strain in 1640 (3), uninoculated 1640 (4), producing strain in MRS (5), non-producing strain in MRS (6), uninoculated MRS (7) and mannose (8) 39
Figure 11.12 Rf values on TLC plates spotted with standards and hydrolyzed recovered polysaccharide after growth of the producing strain of L. plantarum grown on modified MRS. glucose (1), MRS with mannose (2), MRS with lactose (3), MRS with galactose (4), MRS with maltose (5), and mannose (6) 40
Figure 11.13 TLC plate spotted with hydrolyzed and non-hydrolyzed disaccharide, and polysaccharides. Glucose/mannose standard (1:1) (1), hydrolyzed carrageenan (2), hydrolyzed lactose (3), hydrolyzed raffinose (4), hydrolyzed xanthan gum (5), polysaccharide producing strain in 1640 (6), non- polysaccharide producing in 1640 (7), medium 1640 (8). Also included are non-hydrolyzed samples of carrageenan (9), lactose (10), raffinose (11), xanthan gum (12), polysaccharide producing strain (13), non-polysaccharide producing strain (14) and medium 1640 (15) 41
Figure 11.14 Rf values on TLC plates spotted with hydrolyzed and non- hydrolyzed disaccharides, and polysaccharides. Samples include hydrolyzed lactose (1), raffinose (2), xanthan (3,4,5), and carrageenan (6,7). Also included are non-hydrolyzed lactose (8), raffinose (9), xanthan (10) and carrageenan (11) 42 Figure 11.15 TLC plates spotted with glucose (1), hydrolyzed MRS after growth with the non-polysaccharide producing L. plantarum strain in MRS (2), 1:1 glucose and mannose mixture (3), mannose (4), galactose (5), 1:1 gluconic acid and fructose mixture (6), lactose (7), maltose (8), 1:1 glucose and fructose mixture (9), and hydrolyzed MRS after growth with the polysaccharide producing L. plantarum strain (10) 43
Figure 11.16 Medium viscosity after growth of 20 L. plantarum strains in acid whey 44
Figure 11.17 Growth (CFU/mL) of three L. plantarum strains in acid whey supplemented with 2% MRS solution during 4 d of controlled fermentation at 350C and pH 6 45
Figure 11.18 Lactic acid and lactose concentration in acid whey supplemented with 2% MRS solution after 4 d of controlled fermentation with three L. plantarum strains at 350C and pH 6 46 LIST OF TABLES
Page
Table II.l Specific growth rate constant (K) and generation time (g) of polysaccharide producing (335) and non-producing (337) L. plantarum strains grown in MRS 25
Table II.2 Retention time (min) of peaks obtained from hydrolyzed precipitates recovered after growth of the polysaccharide producing L. plantarum strain in modified MRS with various sugars as the sole carbon source 26
Table II.3 HPLC retention time (min) of standard solutions 27
Table 11.4 HPLC retention time (min) of peaks obtained from a 1:1 mixture of internal standard and hydrolyzed precipitate recovered after growth of the polysaccharide producing L. plantarum strain in modified MRS with various sugars as the sole carbon source 28 PRODUCTION AND CHARACTERIZATION OF EXTRACELLULAR
POLYSACCHARIDES PRODUCED BY Lactobacillus plantarum
INTRODUCTION
Whey is the serum or watery part of milk that separates from the curd in the process of making cheese. Whey is produced in large amounts by the cheese industry and, despite decades of research efforts, still causes major pollution problems (Schwartz and Bodie, 1986). From ten pounds of milk there are approximately nine pounds of liquid whey for each pound of cheese produced.
With the increase in environmental concern, whey now has considerable impact on economic and environmental affairs (Gillies, 1974). Whey has a high biological oxygen demand (BOD) exerting approximately 40-50,000 mg BOD/L (Zall, 1979,
1984; Ernstrom, 1986).
Whey disposal became a significant problem when cheese production moved from rural locations to large centralized factories. According to statistics for milk production and processing published by the International Dairy
Federation (IDF), world cheese production in 1983 generated about 1.2 million tons of lactose and 200,000 tons of milk protein of which less than 60% was utilized (Zall, 1984). The rest, which could be a nutritionally valuable food resource was wasted (Kosaric and Asher, 1985). Owing to public concern for the environment and the resulting anti-pollution legislation, the dairy industry must find innovative ways to use whey. 2 Environmentalists look at increased whey production as a potential pollutant of rivers and streams (Gillies, 1974). Humanitarians are concerned that a large percentage of a potential source of excellent protein is wasted (Gillies,
1974). Researchers in universities and industries have therefore looked for new uses for whey and whey derivatives (Zadow, 1984). Cheese manufacturers and municipalities are looking for disposal methods involving the least expense or loss
(Shay and Wegner, 1986). Undoubtedly, the goal should be to identify profitable uses for a product that has been an economic liability for years. Over the past four decades, great efforts have been made to find uses for either whole whey or fractions such as whey proteins. Whey is an important source of protein and contains a balanced spectrum of amino acids and vitamins. Compositional analysis of whey has shown that it contains a significant amount of vitamins and minerals including vitamin A, thiamin, riboflavin, vitamin C, iron, copper and zinc. In general, sweet whey solids (6-6.5% w/w) contains 74% lactose, 13% protein, 0.9% fat, 8.0% ash and 5% moisture. Acid whey solids also at 6-6.5% w/w, comprise
67% lactose, 13% protein, no fat, 10% ash and 3% moisture (Kosaric and Asher,
1985).
The objectives of this study was to evaluate the ability of Lactobacillus plantarum strains for the production of extracellular polysaccharides using acid whey as a growth medium. These polysaccharides can be used in the food industry for their functional properties, particularly as emulsifiers, thickeners and stabilizers and have a high market value. This would generate a new means for acid whey utilization and help eliminate a serious waste disposal problem. LITERATURE REVIEW
Whey as food and beverage ingredient
The use of cheese whey for manufacturing beverages, both alcoholic and nonalcoholic, has been attempted in Europe for many years (Gillies, 1974). Early success (1952) was achieved in developing beverages containing whey constituents.
Rivells is a sparkling crystal clear infusion of herbs in deproteinized whey (Gillies,
1974). In the U.S., efforts have been directed towards whole whey utilization in the form of nutritious beverages, both carbonated and non-carbonated. Among these, citrus flavored beverages, particularly orange, had the highest acceptability
(Gillies, 1974).
The different whey products used as food ingredients include whole whey
(dry and condensed), dry whey fractions, partially delactosed or demineralized whey, protein isolates and concentrates, vitamin B2 and B12 concentrates, and whey vinegar. Whey can be a functional additive or ingredient. Whey protein could serve as a film former in foams and emulsions while whey lactose could be added to pickle brine or used as a carrier in low-calorie sweeteners. Whey is also used in coffee creamers or whiteners. Acid whey can be a source of micronutrients and flavor in the production of fermented products (Zadow, 1984).
Whey is used in infant foods, health foods and imitation milks (Zall, 1984;
Zadow, 1986). One of the main problems when cheese whey is used as a food ingredient is the high mineral content which constitutes about 7% of the total solids. The presence of nitrates, often added during the cheese manufacturing process, causes consumer concerns and thus further limits the use of whey in 4 foods. Whey demineralization, i.e. the removal of 50 to 90-95% of minerals and other ionic species, is already a well established major ingredient in human breast milk substitutes (Milanovic and Caric, 1990). Other applications of demineralized whey include its addition to ice cream, confectionery and bakery products (Jonsson and Olsson, 1981; Potgieter et al., 1986; Rexroat and Bradley, 1986a,b).
Methods specifically designed for the removal of minerals from whey fall into two main categories, i.e. electro-membranes and fixed bed ion exchange resin processes (Evans, 1985). The electro-membrane processes may be divided into four main categories: (a) electrodialysis (ED), (b) transport depletion (TD),
(c) electro-osmosis (EO) and (d) ion substitution (IS). ED and TD are the only electro-membrane processes of commercial importance. A limitation of these processes is the protein content of whey which leads to membrane fouling problems. The fixed bed ion exchange resin processes include conventional ion exchange (IE) and the ammonium bicarbonate process (SMR) (Ahlgren, 1972;
Young, 1974; Delaney, 1976; Houldsworth, 1980; Hill, 1982; Jonsson, 1984).
Whey bioconversions
Bioconversion of food by-products such as whey is one method being considered to increase the value of waste residuals (Moulin and Galzy, 1984).
Whey and whey permeates could be used as a fermentation media for numerous products such as ethanol (Gawel and Kosikowski, 1978; Moulin et al., 1980; Chen and Zall, 1982; Mahmoud and Kosikowski, 1982; King and Zall, 1983a,b; Terrell et al., 1984); single cell protein (Mahmoud and Kosikowski, 1982; Shay and 5 Wegner, 1986; Zall and El-Samragy, 1988); specialized chemicals such as ammoniated organic acid (Gerhardt and Reddy, 1978); lactic acid (Audet et al.,
1989) and hydrolyzed lactose syrups (Patel and Mathur, 1982; Hobman, 1984;
Arndt and Wehling, 1989). Production of ethanol, biomass, ammonium lactate and biogas are all effective treatments in reducing the residual BOD of whey and whey permeate. Maiorella and Castillo (1984) compared the production of ethanol, biomass and lactase production from whey and found that the BOD was reduced by 90-95%. Luksas (1985) has described the production of high quality animal feedstuffs through fermentation of whey by fungi, and the BOD of the effluent was reduced by over 95%.
Alcohol from whey. Alcohol production from whey has received substantial attention especially for incorporation into beverages (Moulin and Galzy, 1984).
A limitation is how to achieve a high percentage of lactose hydrolysis to glucose and galactose which can then serve as substrate for alcohol fermentation since only a few microorganisms are able to carry on this hydrolysis (Thome et al.,
1988). It has been reported that for acid whey concentrated to 10% total solids,
Saccharomyces fragilis is an efficient lactose hydrolyzing strain, however, only 55% of the available lactose was converted, possibly due to the inability of this yeast to tolerate high alcohol concentrations (Kosaric and Asher, 1985).
Single cell protein and biomass. There has been a number of recent studies on the production of single cell protein by fermentation of whey permeate. Castillo 6 and Sanchez (1978) and also Mahmoud and Kosikowski (1982) studied single cell production from concentrated whey permeate by yeast fermentation. This biomass can be a great source of protein especially in underdeveloped countries. However, there are a number of problems associated with single cell production including high nucleic acid content which can lead to gout due to an increase in blood uric acid levels. Consumer attitudes toward the use of single cell protein in food will have to be considered.
Lactic acid production. Lactic acid produced from whey through lactose fermentation can be extensively used in the food industry. It is an established food acid preferred for its bland taste and preserving properties (Lonvand and
Ribereau-Gayon, 1975; Kosaric and Asher, 1985; Roy et al., 1987). Lactic acid uses include beverages and soft drinks, beer manufacturing, acidification of low- alcohol wines, sauerkraut, olives and many dairy products (Kosaric and Asher,
1985).
Microbial solvent production. Several Clostridia strains have been isolated for their ability to grow quickly in batch culture and synthesize solvents from whey permeate. All strains grow on lactose, but solvent production differs widely.
These strains produce a mixture of acetone, butanol and ethanol on glucose- containing media; however, only butanol is produced on acid whey permeate
(Hunter et al., 1990). 7 Microbial polysacchandes
The cell surface of a prokaryote is a unique assembly of polymers, several of which are polysacchandes. A number of extracellular polysaccharides surround the bacterial cell surface. Microbial polysaccharide "gums" are produced during the growth of various genera of bacteria and yeasts. These may take the form of discrete capsules or may be free of any attachment to the cell and are described as slimes (Sutherland, 1977). Many of these gums are useful as thickeners and emulsifiers in a wide variety of industrial applications including foods (Morris,
1984).
Whey can be investigated as a potential medium for the growth of lactobacilli which can ferment lactose directly and tolerate the low pH of acid whey. Many of these microorganisms are capable of synthesizing polysaccharides of interest to the food industry for their use as stabilizers, emulsifiers and for their rheological properties (Glicksman, 1982). Xanthan gum, a polysaccharide produced by Xanthomonas campestris grown in glucose-containing media is being used by the food industry as a stabilizer. Xanthan gum can be produced from media containing deproteinized acid-set and culture-set cottage cheese whey
(Charles and Radjai, 1977). Lactose is hydrolyzed to glucose and galactose prior to fermentation by the yeast. The cost is competitive with glucose media, the process utilizes a high BOD waste stream and it enhances the economics of whey protein recovery. 8 Extracellular polysaccharides. Many bacteria can produce extracellular polysaccharides in the form of capsules surrounding the cell or as slime excreted into the medium (Sutherland, 1977; Roth, 1977; Sand, 1982; Isaac, 1985). Many sugars have been identified as component of extracellular polysaccharides, the simplest being the hexoses D-glucose, D-galactose and D-mannose. Pentoses are comparatively rare. Other neutral sugars found include the 6-deoxyhexoses: L- rhamnose (6-deoxy-L-mannose) and L-fucose (6-deoxy-L-galactose). The common
2-deoxy-2-amino sugars, D-glucosamine and D-galactosamine are found as their
N-acetyl derivatives and many extracellular polymers contain uronic acids. D- glucuronic acid is the most common charged sugar but D-galacturonic, D- mannuronic and L-glucuronic acids are also found (Sutherland, 1977).
Extracellular polysaccharides are usually negatively charged; this is conferred not only by uronic acids but also by non-sugar substituents such as acyl groups, phosphate and ketal substituents (Isaac, 1985). Although many polysaccharides have been studied, rigorous structures have been determined only for a few.
Often only sugars and their approximate molar ratios are known. Furthermore, there is widespread structural variation between polysaccharides even from different strains of the same bacterial genera (Roth, 1977).
Structural analysis of microbial polysaccharides. Many of the polymeric microbial polysaccharides can be characterized by the recently developed techniques of structural polysaccharide chemistry (Sand, 1982). These include chemical and enzymatic methods to degrade the polysaccharide to their 9 constituent oligo- or monosaccharides which are then separated by a range of chromatographic methods including gel filtration, gas liquid chromatography and high performance liquid chromatography. Mass spectroscopy can be used to identify the monosaccharides or their derivatives and H-NMR can be used to determine the anomeric configuration of the glycosidic linkages (Sand, 1982).
Polysaccharides applications in foods. The natural edible gum industry is experiencing a period of growth fueled by a variety of factors including the rapidly increasing interest in low-calorie foods. This has resulted in an expanding need for improved thickeners, stabilizers and emulsifiers (Glicksman and Farkas, 1966).
The greater sophistication of fast production lines requires more stringent control of viscosity during processing. Furthermore, the recognition of gums as "healthy" and the production of new gums by biotechnological processes which are coming into commercialization after years of research and development ensure an even wider application of gums produced by fermentation (Glicksman and Farkas, 1966;
Anonymous, 1989). 10 MATERIALS AND METHODS
Polysaccharide producing bacteria
A polysaccharide producing (335) and a non-producing strain (337) of
Lactobacillus plantarum, originally isolated from a cucumber fermentation, were transferred and maintained by 1% inoculation in MRS broth (Speck, 1984) (Difco,
Inc., Detroit, MI). Test tubes with this broth were incubated at 350C for 24 h and left at room temperature for 6 d until subsequent transfers. Growth rate measurements were conducted at 15, 20, 25, 30,35 and 40oC for both strains using
MRS as growth medium. Inoculation was done at 1% and was followed by incubation at the required temperature with absorbency measured spectrophotometncally at 650 nm during the exponential phase every 30 min
(DMS 80 UV-Visible spectrophotometer, Varian Techtron, Mulgrave, Australia).
Generation time of each strain at above temperatures was calculated using the following equations:
lo<3™N = T^ll + Io9^o (1)
K = slope*2.303 (2)
9 = -^ O) where g is the generation time, K is the specific growth rate constant, and N is
number of cells per unit volume. In some cases optical density (OD) was used 11 instead of N. The slope is calculated from a plot of log10N or log10OD against time (Ingraham et al., 1983).
Culture media and growth of bacteria. Microbial growth and polysaccharide production in media with different sugar sources was evaluated as follows. A modified MRS broth was prepared using bactopeptone 5 g/L, bactotryptone 20 g/L, yeast extract 5 g/L, ammonium citrate 2 g/L, magnesium sulfate 0.1 g/L, manganese sulfate 0.05 g/L, dipotassium phosphate 2 g/L, Twin-80 1 mL/L, and
20 g/L of either glucose, lactose, fructose, galactose, sucrose, mannose, maltose, raffinose, ribose and arabinose (Sigma Chemical Co., St. Louis, MO). The media were sterilized at 1210C for 15 min followed by 1% inoculation of polysaccharide producing and non-producing strains of L. plantarum. Growth was followed by optical density measurements and by total plate counts on MRS. Glucose utilization was determined as follows. One mL samples were removed every hour for a period of 12 h during the exponential phase. Samples were diluted 1:1000, pipetted into colorimetric tubes to which 1 mL of 5% phenol solution was then added. Five mL of concentrated sulfuric acid (98%) was added to each tube, allowed to sit for 10 min, mixed and placed in a 250C water bath for 10 min during which the reaction takes place and color develops (Dubois et al., 1956).
The color of the solutions was measured at 490 nm using a DMS 80 UV-Visible spectrophotometer (Varian Techtron). A standard curve was prepared using
0.001-0.006% glucose solution. The slope and vertical intercept were used to calculate the amount of sugar present in the media. 12 Electron microscopy characterization. MRS was inoculated with the polysaccharide producing and non-producing strains, after 24 h the broth was centrifuged for 20 min at 16,000 g. The pellet was resuspended and washed twice with a 0.85% w/v NaCl solution. Resuspended solution samples were kept for 1 h in a fixative solution (4 mL 25% glutaraldehyde, 10 Ml formaldehyde, 1.6 g
NaH2P04 and 0.27 g NaOH), stained them with phosphotungstic acid and then treated following established procedures (McDowell and Trump, 1976; Wiegel and
Dykstra, 1984). Transmission electron microscopy (TEM) micrographs were prepared at the Electron Microscopy Laboratory of the Veterinary College at
North Carolina State University. Scanning electron microscopy (SEM) micrographs were prepared at the Electron Microscope Service Laboratory at
Oregon State University.
Extraction and puriflcation of polysaccharide. The procedure for the recovery and purification of polysaccharides is summarized in Figure 1.1 (Cerning et al.,
1986; Buller and Voepel, 1990). Late exponential phase cultures were centrifuged using a rotary clinical centrifuge (Sorvall Superspeed RC2-B, DuPont Co.,
Wilmington, DE) at 8000 rpm for 20 min. The precipitate was discarded and the supernatant was concentrated using molecular porous membrane tubing
(Spectra/por, Spectrum Medical Industries) with a molecular weight cut-off of 6-
8000. Polyethylene glycol (PEG, MW 8000, Sigma Co.) was added on the membrane surface. PEG was added for a period of 12-15 h after which the supernatant volume had decreased to one tenth of the original volume. Three 13 volumes of ethanol (95%) and one volume of acetone were added to the concentrated supernatant. Samples were allowed to sit overnight under refrigeration to allow for polysaccharide precipitation. The precipitated polysaccharide was recovered by decanting the supernatant. The precipitated fraction was dissolved in 2-3 mL of distilled water, placed in bags (8000 molecular weight cut off, Spectrum Medical Industries, Inc., Los Angeles, CA) and dialyzed for 24 h against a 2% potassium phosphate (dibasic) solution. Three solution changes were used to remove excess salts, small proteins, and other impurities.
The purified sample was then lyophilized using a bench top freeze drier (Model
75035, Labconco Corp., Kansas City, MO) prior to hydrolysis and chromatography analysis.
Polysaccharide hydrolysis. Ten mg of the recovered lyophilized material was hydrolyzed in 1 mL 2N trifloroacetic acid (99.8%, J. T. Baker, Inc., Phillipsburg,
NJ) using 1 mL teflon bottles (Albersheim, 1967). The hydrolysis was done at
1210C for 1 h in a heating block (cat #13259-005, VWR). The degree of hydrolysis was determined by heating samples 1-4 h at 1210C. After hydrolysis, the solution fraction (TFA) was evaporated to dryness under a stream of filtered air prior to analysis by TLC or HPLC (Honda et al, 1981).
Protein determination. The Lowry assay was used to detect the presence of proteins in the precipitated polysaccharide (Lowry et al., 1951). A calibration curve was prepared by using 0 to 0.1 mL aliquots of bovine serum albumin 14 solution (1.4 mg/mL, Bio-rad Laboratories, Richmond, CA) (Figure 1.2). For protein determination in the purified polysaccharide, 0.1 to 0.3 mL of a 10 mg/mL polysaccharide solution were added to three test tubes. The volume in the sample and standard solution test tubes was adjusted to 0.5 ml using 0.1 N NaOH and mixed vigorously. Five mL of a 1:1 mixture of 2% CuSCVSHjO and 4%
KNaC4H606« 4H20 solution was then added. The tubes with the mixture were allowed to sit at room temperature for 15 min. Thereafter, 0.5 mL of a 1:1 mixture of 2N Folin reagent and water was added to the tubes and allowed to sit for 30 min. Absorbance was measured at 700 nm.
Chromatography. The Shimadzu liquid chromatography system (Shimadzu
Scientific Instruments, Columbia, MO) consisted of a LC-6A solvent delivery unit, a RID-6A refractive index (RI) detector and a CR 501 Chromatopac data processor. The RI detector was used for sugar detection and the analysis was performed isocratically at 0.8 mL/min and 850C using a 300 mm x 7.8 mm i.d. calcium exchange column (HPX-87C; Bio-rad Laboratories) with water as mobile phase.
TLC was performed using as solvent n-butanol, acetic acid (VWR,
Portland, OR) and water in a 4:2:1 ratio. The color reagent was diphenylamine, aniline and phosphoric acid in a 5:5:1 ratio. A Kodak thin layer chromatography sheet (Eastman Kodak Co., Rochester, NY) was used to spot 5 /xL samples. The sheets were allowed to develop for 2 h in the solvent chamber, sprayed with the 15 color reagent, air-dried and then heated in an incubation chamber at 100oC for
10 min (Chrums, 1982).
Viscosity measurements. Rheological properties of cultures in MRS broth were measured over a 24 h period of growth for a polysaccharide producing and a non- producing L. plantarum strain. Viscosity measurements were conducted using a
Cannon-Fenske viscometer (Industrial Research glassware LTD, Union, NJ).
Measurements were conducted on aqueous solutions of the lyophilized precipitate containing the polysaccharide for cultures grown in MRS, a defined media (RPMI-
1640, Sigma Chemical Co., St. Louis, MO) and in acid whey. Ten mL samples were used in tubes with capillary size 50 placed in a water bath at 250C. Samples were allowed to sit in the bath for 10 min to reach temperature equilibration.
Suction was then applied to the tube tip drawing the liquid to the flask mark and the liquid was allowed to flow freely past the second flask mark. The flowing time was measured in seconds and the kinematic viscosity of the samples was calculated in centipoise by multiplying the efflux time in seconds by the viscometer constant.
The viscometer constant was calculated by using the published viscosity value for water at 250C (Weast, 1984) and experimental determinations.
Acid whey fermentation by L. plantarum strains
Growth parameters and polysaccharide production in acid whey were investigated using an additional twenty strains of L. plantarum. Acid whey was obtained from a local cheese processing plant (Sunny Brook Dairy, Corvallis, OR), 16 pH was adjusted from 4.17 to 6.0 using 2N NaOH, filtered to eliminate floating particles and refiltered through a Cellite (Manville Sales Corp., Lompoc, CA) bed applying vacuum to clarify the whey from excess proteins and particles. This solution was filter sterilized using one liter flasks with 0.2 /xm sterile filters (Nalge
Co., Redmond, WA).
Test tube studies. Cultures grown in MRS were used to inoculate (at 1%) test tubes containing acid whey supplemented with 2% MRS broth which was added to provide micronutrients required for bacterial growth. Cultures were incubated at 350C for maximum growth rate. Titratable acidity, expressed as % lactic acid and pH of the cultures were measured after 4 d of acid whey fermentation.
Controlled pH fermentation. Three strains were selected for their ability to increase the viscosity of acid whey after fermentation. Strains 335, 59 and 337 (as control) were inoculated in acid whey sterilized at 1210C for 15 min. These three cultures were inoculated in 2 L sterile acid whey solutions in a custom designed pH and temperature controlled fermenter (Dept. of Microbiology, OSU). The pH of the media was kept constant at pH 6.0 using 1.0 N NaOH to maximize growth and extend lactose fermentation. The temperature of the fermenter was constant at 350C and as in the test tube studies, acid whey was supplemented with 2%
MRS. Cultures were allowed to grow on acid whey for 4 d with samples taken every 12 h for bacterial enumeration as viable cell counts on MRS agar. 17
CULTURE
Filtration or Centrifugation
CONCENTRATED SOLUTION (obtained by membrane dialysis)
ADDITION OF ETHANOL AND ACETONE
PELLET SUPERNATANT -► DISCARD
Purification
PURIFIED POLYSACCHARIDE
lyophilization
LYOPHILIZED POLYSACCHARIDE
Figure I.l Procedure for isolation and purification of polysaccharide 18
Q O
Figure 12 Protein determination by the Lowry assay. Calibration curve of optical density versus BSA (mg) 19 RESULTS AND DISCUSSION
Polysaccharide producing bacteria
The growth rate of polysaccharide producing and non-producing strains of
L. plantarum was evaluated at six temperatures and no difference in growth rate was observed (Figure II. 1). Both strains exhibited a minimum generation time at
350C. Below 20oC growth slowed down significantly and no growth was observed
at 150C and above 40oC. A similar pattern of temperature effect was shown by growth measurements using standard plate counts and spectrophotometric analysis
(Table II. 1). It should be noted that polysaccharide production could have interfered with the spectrophotometric determination of growth. Further studies
are needed to investigate this possibility.
Culture media and growth of bacteria. At 350C both strains grew on modified
MRS with glucose, galactose, sucrose, mannose, fructose, maltose and raffinose as
the sole carbon source. No growth of either strain was observed when ribose and
arabinose were used as the sole sugar substrate.
When MRS was incubated with the polysaccharide producing strain an
increase in medium viscosity was observed, whereas, the non-producing strain
caused no effect. Colony appearances on MRS agar of the polysaccharide
producing and non-producing strains were similar when incubated at 30-40oC;
however, when incubated at 250C for 4 d, the polysaccharide producing strain
formed large, mucoid colonies with shiny surfaces (Figure II.2). The non-
producing strain formed small and non-viscous colonies under the same conditions. 20 Sugar uptake determinations showed that the same amount of glucose in
MRS was fermented by the two strains at a given time (Figure 11.3). Furthermore,
there was no evidence of difference between the two strains in lowering MRS pH
(Figure II.4).
Electron microscopy characterization. TEM micrographs showed slime secretion
and extracellular material attached to the cells of the polysaccharide producing
strain while no such material was evident for the non-producing strain of L. plantarum (Figure 11.5). SEM micrographs of the two strains show that cells of
the polysaccharide producing strain adhered to each other, again reflecting a
difference between strains (Figure II.6).
The viscosity of the cultures in MRS was measured with Cannon-Fenske
capillary viscometers and expressed in centistokes during the growth of both
strains at 350C with uninoculated MRS used as control. The polysaccharide
producing strain increased the medium viscosity 15-25 h after inoculation when
compared to either the non-producing strain or the uninoculated medium;
however, this effect disappeared gradually with incubation time (Figure II.7). One
can speculate the presence of enzymes which can hydrolyze the produced
polysaccharide. A similar result was observed when a simple, defined medium
(RPMI-1640) was used for growth (Figure II.8). This medium was chosen to
eliminate the presence of complex media components that could possibly interfere
with measurements. Glucose at 2% was the sole sugar source and 1% yeast
extract was added to provide micronutrients for growth. Again, the polysaccharide 21 producing strain showed an increase in viscosity 10 h after inoculation which dropped thereafter to values measured for the non-producing strain (Figure II.8).
Polysaccharide production. The polysaccharide-producing strain of L. plantarum was grown in modified MRS with glucose, galactose, mannose, fructose, sucrose, maltose and lactose as sole sugar sources. This was done to detect possible differences in polysaccharide structure as affected by sugar source. Centrifugation to remove cells was followed by precipitation of the polysaccharide with an acetone-alcohol mixture (1:3). The precipitated material was then dialyzed and lyophilized. Analysis of the lyophilized precipitate showed no evidence of protein presence. Hydrolyzed samples were then analyzed by chromatography using several sugars and sugar acids as standards.
The retention time of the peaks obtained from all samples are shown in
Table II.2 while those for standards are listed in Table II.3. Many of the peaks obtained were common regardless of the sugar source (Figures II.9 and 11.10).
The peak at 10 min shows the presence of glucose and a peak at 12 min matches the retention time for mannose. The peak observed at 7-8 min is due to the TFA used to hydrolyse the samples which was not completely removed by air drying.
The peak at 16 min corresponds to the ethanol used for polysaccharide precipitation. When hydrolyzed MRS was injected as control, several peaks matched the retention time of glucose, TFA, ethanol and other unidentified peaks observed in hydrolyzed polysaccharide samples. 22 Hydrolyzed polysaccharide samples isolated after L. plantarum fermentation using various sugar sources were mixed with glucose or galactose standards in a
1:1 ratio. A peak corresponding to galactose was only present when galactose was added to the sample, showing that no galactose is present in the structure of polysaccharide. A glucose peak was present in the chromatogram for hydrolyzed polysaccharide samples with and without glucose as an internal standard. This could indicate that glucose is a component of the polysaccharide structure (Table
II.4). A peak at 12 min was also present in all samples which matches the retention time of mannose.
Thin layer chromatography. In an attempt to characterize the polysaccharide, the lyophilized, hydrolyzed precipitates obtained after growth of the polysaccharide producing L. plantarum strain on MRS and RPMI-1640 media were spotted on
TLC plates along with mannose and glucose as standards (Figure 11.11). Rf values calculated from the plates showed mannose as a single spot with the largest Rf value (0.39). Glucose showed as a single spot with a 0.35 Rf value. The producing and non-producing strains grown on 1640 medium showed one spot with the same Rf value as the uninoculated medium. The Rf values were smaller than those for mannose and glucose. The same results were observed for the hydrolyzed precipitate recovered after growth of both strains on MRS. Again, no significant difference in Rf values were observed including that for the uninoculated MRS control. 23 Samples obtained from the fermentation on modified MRS were also spotted. The modified MRS contained as sugar sources mannose, lactose, galactose, or maltose. Glucose and mannose were used as reference standards.
The Rf values for the single spots observed matched that of mannose (Figure II-
12). This observation was consistent with the HPLC analysis.
The degree of hydrolysis and plate resolution was tested using lactose, raffinose, xanthan gum and carrageenan after hydrolysis following conditions identical to the lyophilized precipitates. Also included were non-hydrolyzed solutions of lactose, raffinose, xanthan gum and carrageenan. Lactose and raffinose when not hydrolyzed traveled only slightly on the TLC plates (Rf values of 0.06) whereas xanthan gum and carrageenan did not travel at all (Figure 11.13 and 11.14). Hydrolyzed lactose and raffinose showed one spot with Rf values of
0.24 and 0.21, respectively. Hydrolyzed xanthan gum showed three distinct spots with Rf values of 0.14, 0.26 and 0.42. Hydrolyzed carrageenan showed two separate spots with Rf values of 0.2 and 0.9. These results indicate that hydrolysis was effective in breaking the polymer units; however, plate resolution was not sufficient to distinguish between monosaccharides obtained from the hydrolysis.
Lactose and raffinose appeared as one single spot on the plate after hydrolysis, however, separation was observed when a 1:1 glucose and mannose mixture was spotted (Figure 11.15). 24 Acid whey fermentation
Twenty different strains of L. plantarum, including the polysaccharide producing strain, were selected for growth and polysaccharide production evaluation on acid whey. Acid whey was filtered, adjusted to pH 6.0, sterilized, and a 2% MRS solution was added to provide micronutrients required for bacterial growth. All strains were incubated at 350C, fermented lactose and grew on acid whey to a maximum of 108 cells/mL within 36 h. A Cannon-Fenske viscometer was used to determine broth viscosity after 20 h of growth with each of the strains tested. Strains 335 (polysaccharide producing strain) and 59 showed a viscosity increase (Figure 11.16). These two strains and the non-polysaccharide producing strain (337) were evaluated for their ability to ferment lactose in acid whey supplemented with 2% MRS broth. A fermenter with pH automatically adjusted to 6.0 and temperature kept constant at 350C was used to follow growth for four days. Samples were taken during the fermentation process for determination of standard plate counts and lactic acid production. Strain 59 showed a maximum growth at 20 h yielding a viable cell count of 109 CFU/mL
(Figure 11.17) and fermented more lactose to lactic acid as compared to the other two strains (Figure 11.18). Following 4 d of acid whey fermentation, the non- polysaccharide producing L. plantarum strain increased lactic acid concentration from 0.05% to 2.1% which represents a reduction in lactose concentration from about 4.6% to 2.65%. The polysaccharide producing strain increased lactic acid to 2.57% while strain 59 increased it to 3.3% which are equivalent to reducing lactose levels to 2.21% and 1.51%, respectively (Figure 11.18). 25 Table II. 1 Specific growth rate constant (K) and generation time (g) of polysaccharide producing (335) and non-producing (337) L. plantarum strains grown in MRS
Strain 337 Strain 335
Temperature K^h"1 g, min fCh"1 g, min oc 20 0.139 298 0.140 297 25 0.207 200 0.214 194 30 0.347 119 0.357 117 35 0.435 95 0.442 94 40 0.196 212 0.219 189 26 Table II.2 Retention time (min) of peaks obtained from hydrolyzed precipitates recovered after growth of the polysaccharide producing L. plantarum strain in modified MRS with various sugars as the sole carbon source
CARBON RETENTION TIME (min) SOURCE fructose 8.1 10 11.1 12.2 16.2 glucose 7.9 8.6 9.4 10.9 12.2 16.3 galactose 7.9 8.7 10.8 12.3 16.3 mannose 8.4 10.6 12.3 16.3 sucrose 7.7 8.5 16.3 maltose 7.9 8.7 9.7 10.8 12.3 16.3 lactose 7.9 8.5 9.1 10.9 12.2 16.3 18.2 27 Table II.3 HPLC retention time (min) of standard solutions
STANDARD RETENTION TIME (min) glucose 10.7 galactose 11.7 mannose 12.2 fructose 13.4 rhamnose 12.3 glucuronic acid 9.5 galacturonic acid 8.4 9.1 mannuronic acid 13.0 gluconic acid 15.6 N-acetyl- 11.4 glucosamine mannose 9.1 12.3 galactose glucuronic acid fructose 10.8 13.4 15.5 glucose gluconic acid 28 Table II.4 HPLC retention time (min) of peaks obtained from a 1:1 mixture of internal standard and hydrolyzed precipitate recovered after growth of the polysaccharide producing L. plantarum strain in modified MRS with various sugars as the sole carbon source.
STANDARD/ RETENTION TIME(min) SAMPLE (# PEAKS) galactose/ 7.7 9.0 10.4 12.3 16.3 mannose gluconic acid/ 7.7 8.4 9.3 10.5 12.3 14.1 16.3 galactose glucose/ 7.6 8.4 9.9 12.3 16.3 glucose glucose/ 7.6 8.4 9.9 12.0 16.3 galactose glucose/ 7.6 8.4 9.9 12.3 16.3 rhamnose glucose/ 7.5 8.5 9.9 12.3 16.3 mannose 29
-0.3 non-producing -0.4 producing -0.5
O -0.6- -0.7- 40
-0.8-
-0.9 0.0031 0.0032 0.0033 0.0034 0.003
1/T (k)
Figure II.l Arrhenius plot for the specific growth rate of polysaccharide producing and non-producing strains of L. plantarum in MRS 30
Figure II.2 Colony appearance of polysaccharide producing (1) and non- producing (2) L. plantarum strains after 5d at 250C incubation in MRS agar 31
non-producing producing U 2- O
o 1-
10 15 TIME (hr)
Figure II.3 Glucose disappearance during growth in MRS of the polysaccharide producing and non-producing L. plantarum strains 32
non-producing producing
X o.
100 200 TIME (hr)
Figure II.4 pH changes during growth of the polysaccharide producing and non- producing L. plantarutn strains in a defined medium (RPMI 1640) supplemented with 2% MRS broth 33
^
Figure II.5 TEM micrographs of L. plantamm a. polysaccharide producing strain b. non-polysaccharide producing strain 34
Figure II.6 SEM micrographs of L. plantarum a. polysaccharide producing strain b. non-polysaccharide producing strain 35
1A broth non-producing producing
1.1 —r~ —r- 20 40 60 80
TIME (hr)
Figure II.7 Changes in MRS viscosity after inoculation with polysaccharide producing and non-producing strains of L. plantarum 36
1.7- non-producing 1.6- u t producing 1.5- >* H (/) 1.4- O U 1.3-
1.2- i.tte&*=e*to*EEs 50 100 150 TIME (hr)
Figure II.8 Changes in the viscosity of a defined media (RPMI 1640) enriched with 2% MRS solution during growth of polysaccharide producing and non-producing L. plantamm strains 37
n
i
Figure II.9 HPLC chromatographs of the recovered precipitate after fermentation with the polysaccharide producing strain of L. plantarum in modified MRS with glucose (1), lactose (2) and sucrose (3) as the sole sugar source 38
"^ -4
Figure 11.10 HPLC chromatographs of the recovered precipitate after fermentation with the polysaccharide producing strain of L. plantarum in modified MRS with galactose (1), fructose (2) and maltose (2) as the sole sugar source 39
> OS
GLUCOSE P+ 1640 P-1640 1640 P+MRS P- MRS MRS MANNOSE
4 5 6 7 8 SAMPLE
Figure 11.11 Rf values on TLC plates spotted with sugar standards, and hydrolyzed recovered polysaccharides after growth of the producing (P+) and non-producing (P-) L. plantarum strains in MRS and 1640 media. Spotting locations correspond to glucose (1), polysaccharide producing strain in 1640 (2), non-producing strain in 1640 (3), uninoculated 1640 (4), producing strain in MRS (5), non-producing strain in MRS (6), uninoculated MRS (7) and mannose (8). 40
SAMPLE
Figure 11.12 Rf values on TIX plates spotted with standards and hydrolyzed recovered polysaccharide after growth of the producing strain of L. plantarum grown on modified MRS. glucose (1), MRS with mannose (2), MRS with lactose (3), MRS with galactose (4), MRS with maltose (5), and mannose (6). 41
8 9 10 11
Figure 11.13 TLC plate spotted with hydrolyzed and non-hydrolyzed disaccharide, and polysaccharides. Glucose/mannose standard (1:1) (1), hydrolyzed carrageenan (2), hydrolyzed lactose (3), hydrolyzed raffinose (4), hydrolyzed xanthan gum (5), polysaccharide producing strain in 1640 (6), non-polysaccharide producing in 1640 (7), medium 1640 (8). Also included are non-hydrolyzed samples of carrageenan (9), lactose (10), raffinose (11), xanthan gum (12), polysaccharide producing strain (13), non-polysaccharide producing strain (14) and medium 1640 (15) 42
LACH RAFFH XAN11 XANH XANTH CARRAH CARRAH LACTNH RAFFNH XANNHCARRANH 123 456789 10 11 SAMPLE
Figure 11.14 Rf values on TIX plates spotted with hydrolyzed and non- hydrolyzed disaccharides, and polysaccharides. Samples include hydrolyzed lactose (1), raffinose (2), xanthan (3,4,5), and carrageenan (6,7). Also included are non-hydrolyzed lactose (8), raffinose (9), xanthan (10) and carrageenan (11). 43
Ji^t'-'V ■;--.V ■ .■>.'-'-':v*"-:'-'i-"'s< ■ ^f^Sn
10
Figure 11.15 TLC plates spotted with glucose (1), hydrolyzed MRS after growth with the non-polysaccharide producing L. plantarum strain in MRS (2), 1:1 glucose and mannose mixture (3), mannose (4), galactose (5), 1:1 gluconic acid and fructose mixture (6), lactose (7), maltose (8), 1:1 glucose and fructose mixture (9), and hydrolyzed MRS after growth with the polysaccharide producing L. plantarum strain (10). 44
a U
8
1 11 12 31 32 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 335 STRAIN
Figure 11.16 Medium viscosity after growth of 20 L. plantarum strains in acid whey adjusted initially to pH 6 and enriched with 2% MRS 45
20 non-producing producing 59 o
-1 B D U
100
TIME (hr)
Figure 11.17 Growth (CFU/mL) of three L. plantarum strains in acid whey supplemented with 2% MRS solution during 4 d of controlled fermentation at 350C and pH 6 46
D u < ui—i H U <
Whey
1/2 O H U <
337 335 STRAIN
Figure 11.18 Lactic acid and lactose concentration in acid whey supplemented with 2% MRS solution after 4 d of controlled fermentation with three L. plantarum strains at 350C and pH 6 47 CONCLUSIONS
Waste management is an important environmental and economic concern
for the food industry. Acid whey, a by-product the from cheese industry, has the
potential to be used as a fermentation medium. However, its usefulness may be
limited because relatively few bacteria can grow on this low pH medium and many
are unable to ferment lactose. In such cases, lactose is hydrolyzed to glucose and
galactose by other methods prior to fermentation.
L. plantarum strain 335 showed evidence of extracellular polysaccharide
production and increased the viscosity of its growth medium. Our major goals were characterization of the bacterium and isolation, purification, identification
of the polysaccharide produced.
MRS medium was used as a model for growth of the bacteria and isolation
of the polysaccharide. An initial observation was that the bacteria produced an
unstable viscous material in the medium. This instability might be due to the
presence of polysaccharide degradative enzymes in the cell, shear stress or other
environmental factors. When a simple defined medium was used, no precipitate
was formed and growth was not optimized. The identification of a growth
medium with all the components required for maximum growth and optimum
polysaccharide production is important. Inadequate amounts limit growth and
affect polysaccharide production while excess nutrients can interfere with
polysaccharide recovery procedures.
Isolation of the polysaccharide was not completely successful due to the
presence of material from the growth medium that co-precipitated with the 48 polymer. An alternative to the solvent precipitation approach used in this study would be an acid or base precipitation which is sometimes useful in the isolation of large polymers.
Presence of excess growth medium components and various reagents introduced during analysis could have interfered with the HPLC analysis. The retention time of many monomers were very close, which led to poor resolution and difficult identification of peaks on the chromatogram. Gas liquid chromatography (GLC) analysis is another technique that could be used as a separation and detection method for the determination of polysaccharide composition.
In conclusion, this study showed that several L. plantarum strains are capable of directly fermenting lactose using it as the sole carbon source for growth. Acid whey can be used as a growth medium and its lactose content can be reduced through fermentation. This reduced-lactose acid whey could be incorporated in various food products.
An increase of medium viscosity was observed when strains L. plantarum
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