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Scientific
Publications - Work Done by Microbiology Reader
Zhennai Yang, Antimicrobial compounds and extracellular polysaccharides produced by lactic acid bacteria structures and properties, Dissertation, University of Helsinki, Faculty of Agriculture and Forestry, Department of Food Technology, March 2000, 61 pp. ABSTRACT Antimicrobial compounds and exopolysaccharides (EPSs) produced by dairy lactic acid bacteria (LAB) were studied. These compounds were separated and purified, and their structures were investigated. The activity of the antimicrobial compounds and the rheological properties of the EPSs were also studied. Thirteen Lactobacillus and five Pediococcus strains were shown to produce a low-molecular mass antimicrobial compound, which was separated and purified by chromatographic methods. Identification by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry showed that the antimicrobial compound was 2-pyrrolidone-5-carboxylic acid (PCA), also known as pyroglutamic acid. The technique of anion exchange chromatography developed in this study was essential for effective separation of PCA from lactic acid, facilitating the identification of PCA. PCA has a wide spectrum of antimicrobial activity against various food-borne spoilage bacteria, including the genera Bacillus, Enterobacter, Escherichia, Klebsiella, and Pseudomonas. The activity of PCA was stable after heat treatments. Compared to lactic acid, PCA had slightly lower antimicrobial activity. On the basis of the results of this study, PCA can be considered, following the identification of reuterin produced by Lb. reuteri, to be another well identified low-molecular-mass antimicrobial compound produced by LAB with a wide spectrum of activity. Lactic acid bacteria are known to produce viscous or non-viscous EPSs. In this study, ten LAB strains have been found to produce EPSs, which have been isolated. Studies on the EPSs produced by Lb. helveticus strains showed that strain Äki4 produced a neutral EPS which was found to be not viscous, whereas strain Lb161 produced a neutral and viscous EPS. Sugar analysis, methylation analysis, and one- and two-dimensional NMR spectroscopy showed that the EPSs produced by Lb. helveticus Äki4 and Lb161 consisted of a hexasaccharide and a heptasaccharide repeating unit, respectively:
Further studies on the EPSs produced by Finnish fermented milk ‘viili’ strains showed that the slime-forming Lactococcus lactis ssp. cremoris strains (ARH53, ARH74, ARH84, ARH87, B30) produced EPSs of relatively high viscosities. These EPSs differed in viscosity in aqueous solutions, and the viscosity was temperature, pH and salt dependent. Addition of EPS to skim milk significantly increased the viscosity, and gelation occurred at 40 °C. 1H NMR spectroscopy showed that the EPSs had a similar or probably identical structure to the one previously reported.
LIST OF PUBLICATIONS This thesis is based on the following publications which are referred to in the text by Roman numerals. Additional data are also presented. I Huttunen, E., Noro, K., and Yang, Z. 1995. Purification and identification of antimicrobial substances produced by two Lactobacillus casei strains. Int. Dairy J. 5, 503-513. II Yang, Z., Suomalainen, T., Mäyrä-Mäkinen, A., and Huttunen, E. 1997. Antimicrobial activity of 2-pyrrolidone-5-carboxylic acid produced by lactic acid bacteria. J. Food Prot. 60, 786-790. III Staaf, M., Widmalm, G., Yang, Z., and Huttunen, E. 1996. Structural elucidation of an extracellular polysaccharide produced by Lactobacillus helveticus. Carbohydr. Res. 291, 155-164. IV Staaf, M., Yang, Z., Huttunen, E., and Widmalm, G. 2000. Structural elucidation of the viscous exopolysaccharide produced by Lactobacillus helveticus Lb161. Carbohydr. Res. (in press). V Yang, Z., Huttunen, E., Staaf, M., Widmalm, G., and Tenhu, H. 1999. Separation, purification and characterization of extracellular polysaccharides produced by slime-forming Lactococcus lactis ssp. cremoris strains. Int. Dairy J. 9, 631-638.
1. INTRODUCTION In a variety of ecological niches, microorganisms compete with each other for survival and through evolution form unique flora. In some food ecosystems, lactic acid bacteria (LAB) constitute the dominant microflora. These organisms are able to produce antimicrobial compounds against competing flora, including food-borne spoilage and pathogenic bacteria (Daeschel 1989, Davidson and Hoover 1993). Under unfavorable environmental conditions many species of LAB also produce exopolysaccharides (EPSs), which protect themselves against desiccation, bacteriophage and protozoan attack (Whitfield 1988, Roberts 1995, Weiner et al. 1995). Lactic acid bacteria provide the major preservative effects in food fermentation which mankind has practiced for thousands of years. The primary antimicrobial effect exerted by LAB is the production of lactic acid and reduction of pH (Daeschel 1989). In addition, LAB produce various antimicrobial compounds, which can be classified as low-molecular-mass (LMM) compounds such as hydrogen peroxide (H2O2), carbon dioxide (CO2), diacetyl (2,3-butanedione), uncharacterized compounds, and high-molecular-mass (HMM) compounds like bacteriocins (Piard and Desmazeaud 1991, 1992, Ouwehand 1998). Among bacteriocins so far characterized, nisin is best defined, and the only purified bacteriocin produced by LAB that has been approved for use in food products (Hansen 1994). A LMM antimicrobial compound, reuterin, has also been chemically identified (Talarico and Dobrogosz 1989), and the reuterin-producing Lactobacillus reuteri strain has been applied as a probiotic in dairy products (Rothschild 1995). The EPSs produced by LAB are either present as a capsule attached to the cell surface, or secreted into the environment (Cerning 1990). Based on their sugar compositions, the EPSs can be divided into homopolysaccharides, composed of a single type of monosaccharide, and heteropolysaccharides, containing several types of monosaccharide (Gruter 1992). Dextran is the most important homopolysaccharide, which is a glucan produced, e.g. by Leuconostoc mesenteroides (Franz 1986). The heteropolysaccharides produced by LAB are generally composed of repeating units of up to eight monosaccharide residues; the chain length and degree of branching vary with the producing strains. The rheological properties of the polysaccharides depend on the monomeric composition, the number of side chains, the chain length and the charge (neutral or anionic) of the polysaccharides, as well as the anomeric configuration of the monosaccharides and the sequence in which they are arranged. The EPSs produced by LAB may act as viscosifying agents to improve the texture and consistency of fermented foods (Cerning 1990, Sikkema and Oba 1998). Since LAB are food-grade microorganisms with the GRAS status (Generally Recognized As Safe), the use of the secreted EPSs as natural alternatives to produce all-natural food products without additives has received increased attention. It has also been claimed that EPSs isolated from LAB cultures have antitumor activity (Oda et al. 1983). Lactic acid bacteria are able to produce a large variety of compounds which contribute to the flavor, color, texture and consistency of fermented foods. However, the present study focuses on two potentially important group of compounds, antimicrobial compounds and EPSs, which differ largely in their chemistry and functionalities. Attention has been paid to developing methods suitable for separation and purification of LMM antimicrobial compounds aiming at inhibition of food-borne spoilage bacteria. In order to understand the relation between the structures and rheological properties of the EPSs, a knowledge of their primary molecular structures is required. In this study, the primary molecular structures of the EPSs produced by Lb. helveticus strains have been studied by NMR spectroscopy. The EPSs produced by several slime-forming Lactococcus lactis ssp. cremoris strains have been characterized to understand the roles of the EPSs in the rheology of fermented foods.
2. LITERATURE REVIEW 2.1. Antimicrobial compounds produced by lactic acid bacteria Lactic acid bacteria are a physiologically diverse group of organisms, which can be generally described as Gram-positive, nonsporing cocci or rods with lactic acid as the major product of carbohydrate fermentation. Traditionally, LAB comprise four genera Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. However, several new genera have been suggested for inclusion in the group of LAB due to a recent taxonomic revision (Axelsson 1998). The genus Streptococcus has been reorganized into Enterococcus, Lactococcus, Streptococcus and Vagococcus. LAB are involved in the fermentation of a range of milk, meat, cereal and vegetable foods (McKay and Baldwin 1990). The antimicrobial compounds produced by LAB can inhibit the growth of pathogenic bacteria of possible contaminants in the fermented products (Raccah et al. 1979, Smith and Palumbo 1983, Cintas et al. 1998). In the past two decades, there have been many reports on the bacteriocins produced by LAB. These bacteriocins are of a proteinaceous nature and they have been grouped into class I, lantibiotics which are small peptides (e.g. nisin), class II, small heat-stable peptides, class III, large heat-labile proteins, and class IV, complex bacteriocins which are not well defined (Klaenhammer 1993). In the following text, the LMM antimicrobial compounds produced by LAB will be discussed.
2.1.1. Organic acids Fermentation by LAB is characterized by the accumulation of organic acids and the accompanying reduction in pH. The levels and types of organic acids produced during the fermentation process depend on the species of organisms, culture composition and growth conditions (Lindgren and Dobrogosz 1990). The antimicrobial effect of organic acids lies in the reduction of pH, as well as the undissociated form of the molecules (Gould 1991, Podolak et al. 1996). It has been proposed that the low external pH causes acidification of the cell cytoplasm, while the undissociated acid, being lipophilic, can diffuse passively across the membrane (Kashket 1987). The undissociated acid acts by collapsing the electrochemical proton gradient, or by altering the cell membrane permeability which results in disruption of substrate transport systems (Smulders et al. 1986, Earnshaw 1992).
Lactic acid is the major metabolite of LAB fermentation where it is in equilibrium with its undissociated and dissociated forms, and the extent of the dissociation depends on pH. At low pH, a large amount of lactic acid is in the undissociated form, and it is toxic to many bacteria, fungi and yeasts. However, different microorganisms vary considerably in their sensitivity to lactic acid. At pH 5.0 lactic acid was inhibitory toward spore-forming bacteria but was ineffective against yeasts and moulds (Woolford 1975). It was possible to grow Aspergillus parasiticus NRRL 2999 in a medium containing 0.5 or 0.75% lactic acid at pH 3.5 or 4.5 (El-Gazzar et al. 1987). Lindgren and Dobrogosz (1990) showed that at different pH ranges the minimum inhibitory concentration (MIC) of the undissociated lactic acid was different against Clostridium tyrobutyricum, Enterobacter sp. and Propionibacterium freudenreichii ssp. shermanii. In addition, the stereoisomers of lactic acid also differ in antimicrobial activity, L-lactic acid being more inhibitory than the D-isomer (Benthin and Villadsen 1995).
Acetic and propionic acids produced by LAB strains through heterofermentative pathways, may interact with cell membranes, and cause intracellular acidification and protein denaturation (Huang et al. 1986). They are more antimicrobially effective than lactic acid due to their higher pKa values (lactic acid 3.08, acetic acid 4.75, and propionic acid 4.87), and higher percent of undissociated acids than lactic acid at a given pH (Earnshaw 1992). Acetic acid was more inhibitory than lactic and citric acids toward Listeria monocytogenes (Ahamad and Marth 1989, Richards et al. 1995), and toward the growth and germination of Bacillus cereus (Wong and Chen 1988). Acetic acid also acted synergistically with lactic acid; lactic acid decreases the pH of the medium, thereby increasing the toxicity of acetic acid (Adams and Hall 1988).
2.1.2. Hydrogen peroxide and carbon dioxide Hydrogen peroxide is produced by LAB in the presence of oxygen as a result of the action of flavoprotein oxidases or nicotinamide adenine hydroxy dinucleotide (NADH) peroxidase. The antimicrobial effect of H2O2 may result from the oxidation of sulfhydryl groups causing denaturing of a number of enzymes, and from the peroxidation of membrane lipids thus the increased membrane permeability (Kong and Davison 1980). H2O2 may also be as a precursor for the production of bactericidal free radicals such as superoxide (O2 -) and hydroxyl (OH.) radicals which can damage DNA (Byczkowski and Gessner 1988).
It has been reported that the production of H2O2 by Lactobacillus and Lactococcus strains inhibited Staphylococcus aureus, Pseudomonas sp. and various psychotrophic microorganisms in foods (Davidson et al. 1983, Cords and Dychdala 1993). In raw milk, H2O2 activates the lactoperoxidase system, producing hypothiocyanate (OSCN-), higher oxyacids (O2SCN- and O3SCN-) and intermediate oxidation products that are inhibitory to a wide spectrum of Gram-positive and Gram-negative bacteria (Reiter and Härnulv 1984, Conner 1993).
Carbon dioxide is mainly produced by heterofermentative LAB. The precise mechanism of its antimicrobial action is still unknown. However, CO2 may play a role in creating an anaerobic environment which inhibits enzymatic decarboxylations, and the accumulation of CO2 in the membrane lipid bilayer may cause a dysfunction in permeability (Eklund 1984). CO2 can effectively inhibit the growth of many food spoilage microorganisms, especially Gram-negative psychrotrophic bacteria (Farber 1991, Hotchkiss 1999). The degree of inhibition by CO2 varies considerably between the organisms. CO2 at 10% could lower the total bacterial counts by 50% (Wagner and Moberg 1989), and at 20-50% it had a strong antifungal activity (Lindgren and Dobrogosz 1990).
2.1.3. Aroma components Diacetyl is produced by strains within all genera of LAB by citrate fermentation. The antimicrobial effect of diacetyl has been known since the 1930s (Jay 1982). It inhibits the growth of Gram-negative bacteria by reacting with the arginine-binding protein, thus affecting the arginine utilization (Jay 1986).
Jay (1982) showed that Gram-negative bacteria were more sensitive to diacetyl than Grampositive bacteria; the former was inhibited by diacetyl at 200 ìg/mL and the latter at 300 ìg/mL. Diacetyl at 344 ìg/mL inhibited strains of Listeria, Salmonella, Yersinia, Escherichia coli, and Aeromonas. Since the production of diacetyl during lactic fermentation is low, e.g. 4 ìg/mL produced by Lc. lactis ssp. diacetylactis (Cogan 1980), and the acceptable sensory levels of diacetyl are at 2-7 ìg/mL (Earnshaw 1992), its practical use as a food preservative is limited. However, diacetyl may act synergistically with other antimicrobial factors (Jay 1992) and contribute to combined preservation systems in fermented foods.
Acetaldehyde is produced by Lb. delbrueckii ssp. bulgaricus by the action of a threonine aldolase, which cleaves threonine into acetaldehyde and glycine. Since Lb. delbrueckii ssp. bulgaricus and S. thermophilus in yoghurt cannot metabolize acetaldehyde, it accumulates in the product at a concentration of about 25 ppm. Acetaldehyde at 10-100 ppm inhibits the growth of Staphylococcus aureus, Salmonella typhimurium and E. coli in dairy products (Piard and Desmazeaud 1991).
2.1.4. Fatty acids Under certain conditions, some lactobacilli and lactococci possessing lipolytic activities may produce significant amounts of fatty acids, e.g. in dry fermented sausage (Sanz et al. 1988) and fermented milk (Rao and Reddy 1984). The antimicrobial activity of fatty acids has been recognized for many years. The unsaturated fatty acids are active against Gram-positive bacteria, and the antifungal activity of fatty acids is dependent on chain length, concentration, and pH of the medium (Gould 1991). The antimicrobial action of fatty acids has been thought to be due to the undissociated molecule, not the anion, since pH had profound effects on their activity, with a more rapid killing effect at lower pH (Kabara 1993).
2.1.5. Reuterin and other low-molecular-mass compounds Reuterin is produced by Lb. reuteri, a heterofermentative species inhabiting the gastrointestinal tract of humans and animals (Axelsson et al. 1987). It is formed during the anaerobic growth of Lb. reuteri by the action of glycerol dehydratase which catalyzes the conversion of glycerol into reuterin (Talarico et al. 1988). Reuterin has been chemically identified to be 3-hydroxypropanal (â- hydroxypropionaldehyde), a highly soluble pH-neutral compound which is in equilibrium with its hydrated monomeric and cyclic dimeric forms (Axelsson et al. 1989, Talarico and Dobrogosz 1989). The biosynthesis pathway from glycerol to the three forms of reuterin is shown below.
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2.3. Methods for evaluation of antimicrobial activity Among many methods available for evaluation of antimicrobial activity (Parish and Davidson 1993), the methods described below have been used for determining the antimicrobial activity of compounds produced by LAB.
2.3.1. The agar diffusion method The agar diffusion method has long been used for testing antimicrobial activity, and it was first used by Fleming in 1924 (Piddock 1990). The method has been widely used for evaluation of antimicrobial activity, specially for biologically derived compounds. It includes agar well diffusion assay and disc assay. In this test, an antimicrobial compound is applied to an agar plate on a paper disc or in a well. The compound diffuses into agar resulting in a concentration gradient that is inversely proportional to the distance from the disc or well. The size of the inhibition zone around the disc or well is a measure of the degree of inhibition. The results of the test are generally qualitative (Parish and Davidson 1993). The method requires that the indicator organisms must grow rapidly, uniformly, and aerobically. Since highly hydrophobic antimicrobial compounds cannot diffuse in agar, they are not suitable for tests by this method (Piddock 1990). Silva et al. (1987) used an agar well diffusion assay for testing the antimicrobial activity of Lb. rhamnosus GG by addition of a 10-fold concentrate of the GG strain or MRS broth to wells (diameter 4 mm) in agar against various anaerobic and facultative bacteria. The activity of an antimicrobial substance produced by Lb. delbrueckii ssp. bugaricus 7994 was tested quantitatively with a disc assay procedure, using paper assay discs 12.7 or 6.35 mm in diameter wetted with 30 or 10 ìl of sample against Pseudomonas fragi and Staphylococcus aureus (Abdel-Bar et al. 1987). The assay methods used for determination of the antimicrobial activity of different species of LAB were slightly different with respect to the sizes of the wells, discs and samples, and the incubation conditions were dependent on the indicator organisms used (Vignolo et al. 1995, Ryan et al. 1996, Choi and Beuchat 1994, Aktypis et al. 1998). Several modified procedures based on the agar diffusion method have also been used for testing antimicrobial activity of LAB. These procedures include the agar spot test (Daeschel and Klaenhammer 1985), deferred antagonism assay (Barefoot and Klaenhammer 1983), and spot-onlawn assay (Hastings and Stiles 1991).
2.3.2. The agar and broth dilution methods Agar and broth dilution methods are used as quantitative methods, suitable for microorganisms with variable growth rate and for anaerobic, microaerophilic microorganisms (Cintas et al. 1995). The results are expressed as MIC, which is the lowest concentration of an antimicrobial that prevents growth of a microorganism after a specific incubation period. In this test, an antimicrobial is serially diluted and a single concentration added to a culture tube or plate added with nonselective broth or melted agar medium, which is then inoculated with test organisms and incubated. The MIC is defined as the lowest concentration at which no growth occurs (absence of turbidity) in a medium following incubation (Parish and Davidson 1993). The broth dilution assay has been used for the determination of the antimicrobial activity of reuterin produced by Lb. reuteri, and the activity of reuterin was expressed as MIC values or as the maximum dilutions of the reuterin fraction (Talarico et al. 1988, Axelsson et al. 1989).
2.3.3. The automated turbidometric assay A turbidometric assay based on automated systems determines the effect of a compound on the growth or death kinetics of a microorganism. It provides information concerning the effect of an antimicrobial that may cause a delayed lag phase or reduced growth rate at concentrations below the MIC. Since the bacterial growth is monitored by measuring the turbidity of the broth medium, the method demands that the instrument be highly sensitive. Growth at levels below log 5.0 CFU/ml may not be detectable (Davidson and Parish 1989). Skyttä and Mattila-Sandholm (1991) described a quantitative method based on automated turbidometry for assaying antimicrobial activity, which was expressed as area reduction percentage values measured under the growth curve. The method has been used to test the antimicrobial activity of antimicrobial compounds produced by P. damnosus and P. pentosaceus (Skyttä et al. 1993) and Lb. plantarum (Niku-Paavola et al. 1999).
2.4. Exopolysaccharides produced by LAB Lactic acid bacteria produce polysaccharides as cell wall components and storage polymers, and also in many species, as a capsule or slime. In the dairy industry, the slime-forming LAB strains have traditionally been used in the production of fermented milk products, e.g. yogurts, Finnish ‘viili’ and Scandinavian ‘långfil’. It has been generally acknowledged that the secreted EPSs by LAB play an important role in the rheological behavior and texture of the products (Sikkema and Oba 1998, De Vuyst and Degeest 1999).
2.4.1. Homopolysaccharides Homopolysaccharides are a group of polysaccharides composed of one monosaccharide type. Several species of LAB are able to utilize sucrose as a specific substrate to produce dextrans, mutans, and levans (Sutherland 1972). Dextrans are a large class of extracellularly formed glucans produced by the genus Lactobacillus, Leuconostoc, and Streptococcus, of which Leuc. mesenteroides and Leuc. dextranicum are the well-known dextran producers. Although each bacterial strain produces a unique glucan, a common structural feature of all dextrans is a high percentage (up to 95%) of á- 1,6 linkages with a smaller proportion of á-1,2, á-1,3, or á-1,4 linkages resulting in a highly branched molecule (Franz 1986). Dextrans are synthesized outside the cell by dextransucrase, which catalyzes sucrose to produce D-fructose and D-glucose, and transfers the latter to an acceptor to form dextran. The reaction is as follows: sucrose + glucan acceptor dextran or mutans + D-fructose Mutans are synthesized in a similar way by S. mutans and S. sobrinus (Montville et al. 1978). However, mutans differ from dextrans in containing a high percentage of á-1,3 linkages, which are attributed to the insoluble nature of this type of polymers (Hamada and Slade 1980). Some S. salivarius strains are able to produce fructans of the levan type with 2,6-linked â- fructofuranoside residues (Cerning 1990). An extracellular enzyme levansucrase is involved in hydrolyzing sucrose and transferring D-fructose to growing fructan chains to form levans: sucrose + fructan acceptor levan + D-glucose
Another type of homopolysaccharide is the galactan produced by Lc. lactis ssp. cremoris H414, which is composed of a branched pentasaccharide repeating unit as shown below (Gruter et al. 1992).
A Pediococcus strain produced a â-D-glucan with a trisaccharide repeating unit (Llaubères et al. 1990). Lactobacillus spp. G-77 has been shown to produce a 2-substituted- (1→3)-â-D-glucan, identical to the EPS produced by P. damnosus 2.6 (Dueñas-Chasco et al. 1997). Lactobacillus spp. G-77 also produced a á-D-glucan composed of a trisaccharide repeating unit (Dueñas-Chasco et al. 1998). Recently, van Geel-Schutten et al. (1998) reported for the first time the production of a fructan by Lb. reuteri strain LB121 with raffinose as a sugar substrate; this strain also produced both a glucan and a fructan on sucrose.
2.4.2. Heteropolysaccharides A wide range of LAB strains can produce heteropolysaccharides, which are composed of repeating units. The monosaccharide compositions of these EPSs are mostly galactose and glucose, and also small amounts of rhamnose, fructose, mannose, and galactosamine (van den Berg et al. 1995). In comparison with the homopolysaccharides, the production of heteropolysaccharides by LAB is much lower (60 to 400 mg L-1) (Stingele et al. 1996). Generally, the heteropolysaccharides are synthesized intracellularly at the cytoplasmic membrane utilizing sugar nucleotides as precursors for the assembly of polysaccharide chains (Cerning 1995). Table 2 lists the heteropolysaccharides produced by the genera Lactobacillus, Lactococcus and Streptococcus.
2.4.2.1. Lactobacillus The ability of lactobacilli to produce EPSs has been recognized for many years. In 1968, Kooiman (from Sikkema and Oba 1998) first reported the structure of a heteropolysaccharide produced by a Lb. brevis strain isolated from kefir grains. This polysaccharide consists of a hexasaccharide repeating unit with D-galactose and D-glucose in the molar ratio 1:1. In the last decade, a number of heteropolysaccharides produced by the Lactobacillus species have been investigated.
Lb. helveticus strains produce several EPSs with varying repeating units, though all containing galactose and glucose (Fig. 1). The EPS produced by Lb. helveticus 776 has hexasaccharide repeating units containing D-galactose and D-glucose (Robijn et al. 1995a). The EPS produced by Lb. helveticus TY1-2 consists of heptasaccharide repeating units with Dgalactopyranosyl and D-glucopyranosyl, and 2-acetamido-2-deoxy-D-glucopyranosyl residues (Yamamoto et al. 1994). Recently, Stingele et al. (1997) showed that the EPS produced by Lb. helveticus Lh59 had an identical primary molecular structure as the one produced by Lb. helveticus TN-4 (Yamamoto et al. 1995), a presumed spontaneous mutant of the strain TY1-2. This polymer is composed of a tetrasaccharide backbone with a lactosyl side-chain, and the molar ratio of D-galactose and D-glucose is 1:1.
Robijn et al. (1995b) reported the primary molecular structure of a viscous EPS produced by Lb. sake 0-1 which was isolated from fermented meat products. The EPS consists of a pentasaccharide repeating unit of glucose, rhamnose, and glycerol phosphate. The threedimensional structure of this polymer has been further studied by molecular mechanics calculations (Robijn et al. 1996b). The helics generated by a polysaccharide builder program are highly extended, with either 2-fold or 3- or 4-fold right-handed chiralities. Grobben et al. (1997) showed that Lb. delbrueckii ssp. bulgaricus NCFB 2772 produced an EPS made up of galactose, and small quantities of glucose and rhamnose, and another EPS that, according to Sikkema and Oba (1998), was similar to the structure of the EPS produced by Lb. delbrueckii ssp. bulgaricus rr (Gruter et al. 1993). The enzymes involved in the production of the sugar nucleotides of strain NCFB 2772 have been analyzed, and based on this analysis a biosynthetic pathway for the EPS has been proposed (Grobben et al. 1996). Growth of the strain in a fructose-based medium led to the absence of the enzyme activities for the synthesis of the rhamnose nucleotide, and accordingly no rhamnose was present in the polysaccharide produced. The EPSs produced by Lb. acidophilus LMG9433 (Robijn et al. 1996c), Lb. kefiranofaciens K1 (Mukai et al. 1990), and Lb. paracasei 34-1 (Robijn et al. 1996a) have also been structurally evaluated, the repeating units being pentamers, hexamers and tetramers, respectively. Recently, Lb. rhamnosus strain C83 has been shown to produce an EPS composed of a pentasaccharide repeating unit with a linear structure (Vanhaverbeke et al. 1998). This strain, as well as Lb. casei CG11 (Cerning et al. 1994) and Lb. sake 0-1 (van den Berg et al. 1995), produced more EPSs at lower temperatures, whereas several other Lactobacillus strains produced more EPSs at higher temperatures (compared with the optimum temperatures of growth) (Grobben et al. 1995, Garcia-Garibay and Marshall 1991).
2.4.2.2. Lactococcus Among lactococci, only the slime-forming Lc. lactis ssp. cremoris strains have been investigated. These strains producing EPSs play a role in the proper consistency of the fermented milk (Cerning 1995). The sugar components of the EPSs are most frequently galactose, glucose, and very often rhamnose (Cerning 1990). Nakajima et al. (1992a) reported a phosphate-containing heteropolysaccharide, named ‘viilian’, produced by Lc. lactis ssp. cremoris SBT 0495 which was isolated from a Finnish ‘viili’ starter culture. The EPS consists of the following repeating unit: Lc. lactis ssp. cremoris B40 has also been found to produce a phosphopolysaccharide with an identical repeating unit as shown above (van Casteren et al. 1998). Lc. lactis ssp. cremoris strain LC330 appeared to produce concurrently two EPSs; an anionic EPS composed of galactose, glucose, rhamnose, glucosamine and phosphate, and a neutral EPS containing galactose, glucose and glucosamine with branched terminal galactose moieties (Marshall et al. 1995). The mechanism for the biosynthesis of the EPS by Lc. lactis ssp. cremoris has not been investigated in detail. Recently, Oba et al. (1996) proposed a biosynthetic pathway for the production of ‘viilian’ by strain SBT 0495 with the following steps: preparation of the membraneembedded lipid carrier; incorporation of the first monosaccharide with the phosphate on C-1; assembly of the intact repeating unit. The biosynthesis of the EPSs produced by Lc. lactis strains is generally associated with a plasmid (Kleerebezem 1999). Transferring mucoidity plasmids from Lc. lactis ssp. cremoris ARH87 and MS to nonmucoid Lc. lactis strains proved the latter to be mucoid (Vedamuthu and Neville 1986, von Wright and Tynkkynen 1987). van Kranenburg et al. (1997) described a novel 12 kb EPS gene cluster located on a 40 kb plasmid, which was essential for the EPS synthesis of Lc. lactis ssp. cremoris NIZO B40. Introduction of the EPS gene cluster from S. thermophilus Sfi6 to a non-EPS-producing Lc. lactis MG1363 produced an EPS with a different structure from the EPS of the native host (Stingele et al. 1999). The absence of the GalNAc residue in the EPS of Lc. lactis MG1363 was probably caused by the lack of a UDP-N-acetylglucosamine C4- epimerase activity (Fig. 2).
2.4.2.3. Streptococcus S. thermophilus strains are used in combination with Lb. delbrueckii ssp. bulgaricus strains in yoghurt starters. The EPSs produced by several S. thermophilus strains have been found to have similar or identical primary molecular structures. Doco et al. (1990) first reported the structure of an EPS produced by ropy S. thermophilus strains CNCMI 733, CNCMI 734 and CNCMI 735, which consisted of a tetrasaccharide repeating unit of D-galactose, D-glucose, and N-acetyl-D-galactosamine in a molar ratio 2:1:1. An EPS with an identical repeating unit structure has been reported to be produced by S. thermophilus Sfi6 (Stingele et al. 1996). Lemoine et al. (1997) showed that the EPSs produced by S. thermophilus Sfi12 and Sfi39 had molecular masses greater than 2 x 106, and both yielded a slimy texture rather than a thickened one in yoghurt. However, they had different sugar compositions and structures; the former consisting of a hexasaccharide repeating unit of galactose, glucose and rhamnose, and the latter a tetrasaccharide repeating unit of galactose and glucose.
Recently, Faber et al. (1998) showed that S. thermophilus Rs and Sts produced EPSs of identical repeating units, but they had different molecular masses, resulting in a difference in viscosity in their milk cultures. Bubb et al. (1997) also showed that the EPS produced by S. thermophilus OR 901 had a similar repeating unit to the one of the strains Rs and Sts; all being branched heptasaccharide repeating units of D-galactose and L-rhamnose in the same molar ratio: 5:2.
2.5. Isolation and structural elucidation of EPSs 2.5.1. Isolation of EPSs EPSs produced by LAB can be isolated by precipitation with trichloroacetic acid (TCA) (Gruter et al. 1992) to remove proteins from the culture media, and subsequently by ethanol (Faber et al. 1998) and/or acetone (Lemoine et al. 1997) to precipitate polysaccharides. Gel filtration or ion exchange chromatogrphy is often used for further purification of EPSs. Robijn et al. (1995a, 1995b, 1996a, 1996b) purified EPSs produced by several Lactobacillus strains by gel filtration with different columns (Sephacel 500, Sephacryl S-500, and Superrose-6). Gel filtration techniques were also used for purification of the EPSs produced by Lc. lactis ssp. cremoris H414 (Gruter et al. 1992), S. thermophilus Sfi16 (Stingele et al. 1996), and S. thermophilus CNCMI 733 (Doco et al. 1990). Since many EPSs are negatively charged, they can be bound to an anion exchanger. This technique has been used for purification of the anionic EPSs produced by Lc. lactis ssp. cremoris B40 (van Casteren et al. 1998), and Lb. helveticus TY1-2 (Yamamoto et al. 1994) and TN-4 (Yamamoto et al. 1995). Marshall et al. (1995) showed that Lc. lactis ssp. cremoris strain LC330 produced at the same time both neutral and anionic EPSs in the medium; these two EPSs were effectively separated and purified by anion exchange chromatography.
2.5.2. Sugar and methylation analyses In the analysis of monosaccharide compositions of EPSs produced by LAB, the EPSs are hydrolyzed with trifluoroacetic acid (TFA), reduced and acetylated, and the acetate derivatives are analyzed with GC. For the methylation analysis, monosaccharides are usually derivatized into partially methylated alditol acetates, which are introduced into the EI source of MS from a GC, or GLC interface. The substitution pattern of the monosaccharides can be determined by comparing their fragmentation pattern with reference EI-MS spectra. Yamamoto et al. (1994, 1995) studied the substitution pattern of the monosaccharides in the EPSs produced Lb. helveticus TY1-2 and TN-4 by GLC-MS of the methylated and acetylated sugar residues. The EPS produced by Lb. helveticus 766 was analyzed by GLC-MS on DB-1 of the partially methylated alditol acetates (Robijn et al. 1995a).
2.5.3. Nuclear magnetic resonance spectroscopy NMR spectroscopy relies on the interaction of radio-frequency electromagnetic radiation with magnetically active nuclei in a strong magnetic field. The radio frequencies used range from 200 to 800 MHz, corresponding to magnetic fields from 4.7 to 18.8 Tesla. 1H and 13C are the spin-active nuclei most frequently encountered in carbohydrates. 1H and 13C NMR spectroscopy, including one- (1D) and two-dimensional (2D), is a powerful tool for structural studies of carbohydrates (Widmalm 1998), which also include polysaccharides produced by LAB. 1D 1H NMR spectroscopy can be used for rapid identification or to check the purity of a polysaccharide sample. Signals in the anomeric region (about 4.3-5.5 ppm) of the spectrum and the coupling of the anomeric protons (JH1,H2) may provide useful information about the number of residues in a repeating unit, and the anomeric configuration, respectively. Yamamoto et al. (1995) recorded the 500 MHz 1H NMR spectrum of the EPS produced by Lb. helveticus TN-4 in D2O at 70 oC, and found six signals in the anomeric region with nearly equal integrated intensities, suggesting there was a hexasaccharide repeating unit for this EPS. A study on the EPS produced by Lb. helveticus Lh59, by 400 MHz 1H NMR spectroscopy in Me2SO-d6 at 80 oC produced four anomeric protons signals in a molar ratio 1:2:2:1, which also indicated a hexasaccharide repeating unit (Stingele et al. 1997). The 1H NMR spectrum of the EPS produced by Lb. paracasei had four doublets in the anomeric region, and the coupling constants (JH1,H2) of these signals (8.3 Hz, 7.7 Hz, 7.3 Hz, 7.7 Hz) were of the pyranoid ring form with all the residues in the â configuration (Robijn et al. 1996a).
Since the natural abundance of 13C is very low (1.1% relative to 12C), the peak intensity of 13C has to be enhanced in 1D 13C NMR spectroscopy by using a large number of pulses, by taking advantage of the nuclear Overhauser effect (NOE), or by using distortionless enhancement by polarization transfer (DEPT) experiments. The values of 13C chemical shift and 13C-1H coupling (1JC,H) provide structural information of the polysaccharides. Robijn et al. (1995a) found six signals in the anomeric region of the 13C NMR spectrum of the EPS produced by Lb. helveticus 766, confirming the suggested hexasaccharide repeating unit by 1H NMR spectroscopy. Based on the C-1 chemical shifts in the 13C NMR spectrum of the EPS produced by Lb. rhamnosus strain C83, Vanhaverbeke et al. (1998) assigned two downfield signals (ä 110.12, 107.67) to two residues having â configuration, and three signals at ä 99.73, 99.99 and 100.73 to three residues having á configuration.
The 1D NMR techniques are often used for the assignment of signals in the anomeric region. For detailed assignment for the spin system of sugar residues, 2D techniques are needed. These techniques include 1H,1H-correlated spectroscopy (COSY), total correlation spectroscopy (TOCSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), gradient selected heteronuclear single quantum coherence (gHSQC), gradient selected heteronuclear multiple-bond correlation (gHMBC) experiment, and nuclear Overhauser effect spectroscopy (NOESY). Bubb et al. (1997) used a TOCSY experiment to further assign two signals of anomeric protons in the 1H NMR spectrum of the EPS produced by S. thermophilus OR 901. By means of 2D COSY, HOHAHA, NOESY, and 13C-1H HMQC (heteronuclear multiple quantum coherence), Robijn et al. (1996a) assigned almost all 1H and 13C resonances in 1D 1H and 13C NMR spectra of the EPS produced by Lb. paracasei 34-1.
The sequence of the monosaccharide residues in a repeating unit can be established by 2D NOESY and HMBC experiments. The former experiment gives information about the interresidue linkage from observation of the NOE between anomeric protons and the protons at the substituted positions of neighbouring sugar residues. The latter experiment gives rise to crosspeak between proton and carbon atoms that are long-range scalar coupled. Faber et al. (1998) used 2D NOESY together with HSQC-NOE experiments to determine the sequence of the sugar residues in the EPS produced by S. thermophilus Rs and Sts. The monosaccharide sequence in the EPS produced by Lb. paracasei 34-1 was established by 2D NOESY experiments (Robijn et al. 1996a).
2.6. Rheological characterization of EPSs 2.6.1. Solution viscosity Viscosity ç is defined as the ratio between shear stress and shear rate. The intrinsic viscosity [ç], which measures the hydrodynamic volume of a molecule, is obtained by extrapolating the Huggins equation to zero concentration: çsp /c = [ç] + k’[ç]2c, where çsp is specific viscosity, c is polymer concentration, and k’ is a constant for a series of polymers of different molecular mass in a given solvent. çsp /c is also defined as the reduced viscosity çred. For ionic polysaccharides in aqueous solutions, the value of çred increases with decreasing concentration, showing a polyelectrolyte effect. The behavior of the polyelectrolytes is influenced by intrachain Coulombic interaction, ionic strength, pH and specific counterions (Paoletti 1998). Oba et al. (1999) suggested that in a strain sweep test at very high dilution of the EPS produced by Lc. lactic ssp. cremoris SBT 0495, the higher cross-over frequency of the EPS in 0.1 M NaCl compared to that in pure water was due to the polyelectrolyte effect of this EPS. van den Berg et al. (1995) showed that over a wide range of shear rates, the viscosity of a 1% solution of the EPS produced by Lb. sake 0-1 decreased with increasing shear rates, indicating a shear-thinning behavior, and the viscosity was comparable to that of xanthan gum.
2.6.2. Dynamic viscoelasticity In response to an applied stress, polysacharides may show a viscoelastic behavior, i.e. a combination of truely viscous flow and perfectly elastic response. In a dynamic test, the polysaccharide sample is subject to sinusoidal shear oscillation with a wide range of frequencies (0.01-300 Hz). The relative magnitudes of G' (storage modulus) and G" (loss modulus) vary with the state of the polysaccharide. For entangled solutions, where there is a greater contribution from the viscous element, G' is low. When frequency decreases, there is a crossover in G' and G", and they flow as high viscosity liquids at very low frequencies. For gel systems, G' and G" are parallel, with G' > G" and largely frequency independent (Ross-Murphy 1998). Oba et al. (1999) showed that in a dynamic and steady shear measurement the aqueous solutions of the EPS produced by Lc. lactis ssp. cremoris SBT 0495 behaved as an entangled solution but not as a weak gel. Nishinari (1997) reported the frequency (0.01 - 10 ù/rad⋅s-1) dependence of G' and G" for 1-3% solutions of gellan gum, an EPS produced by Pseudomonas elodea. The 1% (0-30 °C) and 2% (30 °C) solutions had a typical dilute solution behavior with G" > G'. The 2% (15 °C, 25 °C) and 3% (30 °C) solutions, however, had a concentrated solution behavior with a crossover of G' and G" and G' > G" at higher frequencies. Gelation occurs at 3% at 0-25 °C with G' and G" being slightly frequency dependent.
2.6.3. Gelling properties Gels are defined as loose three-dimensional networks with structures ranging from homogeneous solutions (enthalphy-driven aggregations) to heterogeneous rigid porous systems (Li et al. 1996). Clark and Farrer (1995) described the mechanisms of gel formation in three main classes, firstly by point crosslinking with covalent bonds, secondly by chain association driven by changes in temperature, pH and ionic strength, and the presence of small molecules and specific counterions, and thirdly by particle aggregation. Many polysaccharide gels are formed by thermoreversable physical associations, involving Coulombic, dipole-dipole, van der Waals, charge transfer, and hydrophobic and hydrogen bonding interactions, as well as double-helix formation and aggregation ( Guenet 1992). Gels can be characterized to be strong or weak based on their response to deformation. At large deformations, strong gels will rupture and fail, while weak gels flow without fracture, and show recovery of solid character. Xanthan gum, the EPS produced by Xanthomonas campestris, forms a weak gel, with large deformation fluid properties, but it also forms strong gel under extreme conditions (Ross-Murphy 1995).
2.6.4. Rheological behaviors of EPSs in fermented milk The use of EPS-producing LAB strains may improve the rheological properties of fermented milk. The gel structure and viscosity of the products are affected by the gel formation conditions, as well as the amount and the type of the EPSs produced. Hammelehle et al. (1998) showed that fast warming rates (20-50 °C) during acidification increased the gel firmness and storage modulus, and decreased the syneresis of a milk gel. Skim milk fermented by ropy EPS-producing strains exhibited similar rheological properties, and had greater viscosity than skim milk fermented by non-ropy strains (Schellhaass and Morris 1985). Ropy EPS-producing strains also increased the viscosity of yoghurt when compared to yoghurt made with non-ropy cultures (Rawson and Marshall 1997, Sebastiani and Zelger 1998), and improved the texture of quarg (Sebastiani et al. 1997). As described ealier, S. thermophilus Rs and Sts produced EPSs of the same structure, but had different viscosities in the milk cultures due to their different molecular masses (Faber et al. 1998). The rheological behavior of the polysaccharides is also related to their three-dimensional structure (Robijn et al. 1996b). In addition to the viscosifying effect of the polysaccharides, the interactions between the EPSs and the milk proteins, e.g. caseins, also play a role. Studies of a yogurt gel with a scanning electron microscopy showed that the cells were attached to the protein coagulates by a network structure consisting of polysaccharide filaments (Schellhaass and Morris 1985, Toba et al. 1990). The microorganisms and/or the EPSs that they produce may affect the protein aggregation, thereby affecting the physical properties of the milk gel (van Marle and Zoon 1995). A recent study showed that the rheological properties of stirred yoghurt were affected by the type of EPSproducing strains used, suggesting an effect due to the interaction between the polymer and milk proteins (Marshall and Rawson 1999). Hess et al. (1997) proposed a model for shear-induced degradation of the microstructure of EPS-producing yogurt. Since the associations of EPS with bacterial cells or casein micelles are stronger than the associations between the casein micelles, an increase in shear will first disrupt the casein micelle network that is not associated with EPS, subsequentely the associations between the cells and EPS, and then the portion of the casein network that is associated with EPS.
2.7. Applications in foods and health aspects 2.7.1. Antimicrobial compounds as natural food preservatives The quality of most foods deteriorates during storage. In addition to physical, chemical and enzymatic factors which may alter the sensory characteristics, the microbiological changes in foods may bring about a wide range of spoilage reactions, including food poisoning (Gould 1991). Therefore, it is of significance to inhibit the growth of spoilage microorganisms in foods. Due to a strong demand for natural and minimally processed foods, there has been a growing interest in the use of antimicrobial compounds produced by LAB as a safe and natural way of food preservation.
In addition to nisin which has been widely used in foods (Qiao 1996), another antimicrobial compound that has been proposed for use in food preservation is reuterin produced by Lb. reuteri (Lindgren and Dobrogosz 1990). Addition of reuterin to ground beef was found to inhibit the growth of E. coli. (Daeschel 1989). Surface treatment of herring with a mixture of Lb. reuteri and glycerol significantly improved the shelf-life of the product (Lindgren and Dobrogosz 1990). Lb. reuteri has been commercially used in combination with Bifidobacterium infantis and Lb. acidophilus in sweet and fermented milk under the trade name BRA-mjölk (Rothschild 1995).
Antimicrobial compounds can be applied to foods either as purified chemical agents, or as viable cultures in the case of fermented products (Barnby-Smith 1992). Novel purified antimicrobial compounds require data to substantiate their lack of toxicity in order to obtain approval for their use in foods. Traditional fermented products that naturally contain antimicrobial compounds have been consumed for centuries, and starter cultures with selected antimicrobial properties may be used to replace those used in traditional fermented foods. However, problems may arise with respect to retaining the flavor and texture of the products (Earnshaw 1992).
2.7.2. EPSs in food applications Polysaccharides may function in foods as viscosifying agents, stabilizers, emulsifiers, gelling agents, or water-binding agents (van den Berg et al. 1995). The majority of the polysaccharides used in foods are of plant origin. Most of them are chemically or enzymatically modified in order to improve their rheological properties, e.g. cellulose, starch, pectin, alginate and carrageenan. Therefore, their use is strongly restricted. EPSs of microbial origin have unique rheological properties because of their capability of forming very viscous solutions at low concentrations and their pseudoplastic nature (Becker et al. 1998). The EPSs produced by foodgrade LAB have been considered as a new generation of food thickeners to improve the rheological properties of foods (Robijn 1996). Dextran is the first industrial polysaccharide produced by LAB. It was discovered in 1880 in sugar cane or beet syrups where dextran was found to be responsible for the thickening and gelation of the syrups (Crescenzi 1995). Due to their structural differences, some dextrans are water soluble and others are insoluble. Dextran can be used in confectionary to improve moisture retention, viscosity and inhibit sugar crystallization. In gum and jelly candies it acts as a gelling agents. In ice cream it acts as a crystallization inhibitor, and in pudding mixes it provides the desirable body and mouth feel (Whistler and Daniel 1990). In addition, dextran has also been used as blood plasma extenders and as the basic component of many chromatographic stationary phases (Franz 1986).
Xanthan gum is the second microbial EPS which was approved for use in foods in 1969. Although it is produced by the plant-pathogen Xanthomonas campetris, Sutherland (1998) described xanthan as the "benchmark" product with respect to its importance in both food and nonfood applications, which include dairy products, drinks, confectionary, dressing, bakery products, syrups and pet foods, as well as the oil, pharmaceutical, cosmetic, paper, paint and textile industries. The production of xanthan is relatively inexpensive because of the high conversion of substrate (glucose) to polymer (60-70%) (Sutherland 1998). According to Becker et al. (1998), xanthan in solutions exhibits a high viscosity at low concentrations and strong pseudoplasticity, and it is stable over a wide range of pHs, temperatures and ionic strengths.
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Another probiotic Lb. reuteri also produced an antimicrobial compound with a wide spectrum of activities (see 2.1.5.). Studies on bacterial adhesion showed that capsular polysaccharide might promote the adherence of bacteria to biological surfaces, thereby facilitating the colonization of various ecological niches (Costerton et al. 1987). The EPSs were found to be present in adherent biofilms (Whitfield 1988); the EPSs might function as initial adhesion, and permanent adhesion compounds (Allison and Sutherland 1987). As well as live bacteria (probiotics) which can improve intestinal balance to promote health, dietary carbohydrates may function as prebiotics, beneficially affecting the colonic microflora (Salminen et al. 1998b). These dietary carbohydrates include polysaccharides of plant origin (resistant starch, â-glucan, cellulose, inulin), oligosaccharides (fructo-, gluco-, malto-, xylo- and soybean oligosaccharides), and lactose derivatives (Kontula 1999). There have been no reports of the use of EPSs produced by LAB as prebiotics. Although milk fermented with an EPS-producing strain Lc. lactis ssp. cremoris SBT0495 had cholesterol lowering activity, the mechanism is unknown (Nakajima et al. 1992b).
Oda et al. (1983) reported an antitumor EPS produced by Lb. helveticus ssp. jugurti. The antitumor activity of the EPS was tested against ascites Sarcoma-180 by injecting the EPS preparation intraperitoneally. Mice given a 20 mg kg-1 dose for nine succesive days had an increased life span value of 144%, and a value of greater than 233% corresponding to a 40 or 80 mg kg-1 dose. The authors concluded that the antitumor activity of the EPS might be based on its host-mediated actions. In order to understand the antitumor activity, the effect of the EPSs or the EPS-producing cells on the immune system has been investigated. Forsén et al. (1987) showed that cell surface materials, possibly lipoteichoic acids, of Lc. lactis ssp. cremoris T5 produced Tcell mitogenic activity in human lymphocytes. The slime produced by Bifidobacterium adolescentis had immunomodifying effects on mouse splennocytes (Gómez et al. 1988). Kitazawa et al. (1992) showed that the slime-forming Lc. lactis ssp. cremoris KVS20 had antitumor activity, and the slime contained strong B-cell dependent mitogenic substances.
3. AIMS OF THE STUDY One of the aims was to study the antimicrobial compounds produced by dairy lactic acid bacteria, particularly the low-molecular-mass compound inhibitory toward various spoilage and pathogenic bacteria in foods. Another aim was to study the extracellular polysaccharides produced by dairy lactic acid bacteria in view of the role of the exopolysaccharides in the improvement of the texture and consistency of fermented foods. The specific aims of the study were:
1. To separate, purify and identify low-molecular-mass antimicrobial compounds produced by the lactic acid bacterial strains, and to study the antimicrobial properties of these compounds.
2. To isolate exopolysaccharides produced by the lactic acid bacterial strains, to evaluate the primary molecular structures of the exopolysaccharides, and to study the rheological properties of the viscous exopolysaccharides.
4. MATERIALS AND METHODS 4.1. Antimicrobial compounds produced by LAB (I, II) 4.1.1. Bacterial strains and growth conditions All bacterial strains used in the study of antimicrobial compounds were obtained from Valio Ltd, Research and Development Service, Helsinki, Finland. The bacterial cultures were maintained at -80 °C in glass beads and they were subcultured twice before use. The LAB strains examined for producing antimicrobial compounds were grown in MRS, KCA, and whey permeate or whey media, and incubated at 30 °C or 37 °C (Table 3). Food spoilage bacteria and also LAB strains (Table I/1, Table II/2) were used as indicator organisms for antimicrobial tests.
Table 3. List of lactic acid bacterial strains examined for producing antimicrobial compounds, their growth media and incubation conditions used in this study Strain Growth medium Incubation condition Lactobacillus sp. acidophilus "NCFB" Lb 1748 MRS 30 °C, 25 h casei C MRS 30 °C, 25 h casei ssp. casei LC-10 MRS 37 °C, 72 h casei LC1/6-1 MRS 30 °C, 25 h casei SHIROTA MRS 37 °C, 25 h delbrueckii ssp. delbrueckii strain 13S MRS 37 °C, 25 h helveticus Äki4 MRS 37 °C, 45 h lactis KKNO 1134 Lb78 MRS 37 °C, 25 h lactis ssp. bulgaricus KKNO 312 Lb389 MRS 37 °C, 25 h paracasei ssp. paracasei Lb1931 MRS 37 °C, 72 h reuteri DSM20016 MRS 30 °C, 25 h rhamnosus GG MRS 37 °C, 24 h rhamnosus LC-705 Whey permeate 30 °C, 48 h Lactococcus lactis ssp. diacetylactis EM1* KCA** 30 °C, 24 h Pediococcus sp. strains VN13, VN18, 435, 4025, 4035 MRS 30 °C, 24 h Streptococcus thermophilus T101/85* Whey medium 37 °C, 18 h * Unpublished. * * KCA: calcium citrate agar. 4.1.2. Separation and purification of antimicrobial compounds After the growth of the LAB strains under proper conditions (Table 3), cells in the culture broth were filtered, and the cell-free broth was concentrated 10-fold by lyophilization. The concentrate was then precipitated stepwise by ethanol from 30 to 97.5% with intermediate centrifugation (30 min, 22 000g, 4 °C). The precipitates obtained from each addition of ethanol and/or the final supernatants showing antimicrobial activity were further purified by chromatographic methods.
Gel filtration was performed using a Bio-Rad Econo System (Richmond, CA, USA). The sample (100 mg) was loaded onto a column (75 x 1.5 cm) on Bio-Gel P-2 polyacrylamide gel (M=100-1800, -400 mesh, Bio-Rad) eluted with 0.05 M ammonium acetate (NH4OAc) at a flow rate of 10 ml h-1, and the eluant was monitored at 280 nm. The active fractions were collected, lyophilized, and subjected to anion exchange chromatography using a Bio-Rad Econo system with a column (25 x 1.5 cm) on weakly basic Fractogel TSK DEAE-650(S) gel (Merck, Darmstadt, Germany). Elution was carried out at a flow rate of 1.0 ml min-1 using a stepwise elution program: fractions 1-30 with water; fractions 31-65 with 0.04 M NH4OAc adjusted to pH 5.5 with acetic acid (AcOH); fractions 66-90 with 0.5 M NH4OAc adjusted to pH 5.5 with AcOH. A fraction was collected every four minutes with monitoring at 254 nm. The active fractions (except fractions containing lactic acid) from anion exchange chromatography were further purified by RP-HPLC using a model 600 E multisolvent delivery system equipped with a Baseline 810 software (Millipore Co., Milford, MA, USA). The mobile phase, 0.02 M NH4OAc containing 1% AcOH (pH 3.80), was used after filtration through a membrane filter (pore size, 0.2 ìm). Elution was performed isocratically from a Spherisorb S5 C8 column (250 x 4.6 mm, Phase Separations Ltd, Chester, England), fitted with a C8 precolumn (Millipore) at a flow rate of 0.75 ml min-1, and at 40 °C for 30 min. A fraction was collected each minute. The absorbance was monitored at a range of wavelengths from 190 to 300 nm at an interval of 5 or 10 nm.
4.1.3. Identification of antimicrobial compounds 1H and 13C NMR measurements were carried out on a Bruker AM 400 WB spectrometer (Karlsruhe, Germany), operating at 400.1 MHz for 1H. Spectra were recorded with sample solutions in H2O/D2O (90/10) at ambient temperature and referenced to sodium 3-trimethylsilyl- [2,2,3,3-2H4]propanoate.
The electron impact (EI) and fast atom bombardment (FAB) mass spectra were recorded on a Jeol SX-102 double-focusing spectrometer (Tokyo, Japan).
EI: The sample was injected into the direct probe and the solvent (water) evaporated. The probe was inserted into the ion source (250 °C). The filament was heated at a rate of 16 °C/min up to 300 °C/min, the ionization current being 300 mA. The ionization energy was 70 eV and the accelerating voltage 10 kV. The spectra were recorded over the range 10-500 m/z. Calibration was based on PFK (perfluorokerosin, positive ion mode).
FAB: The sample was introduced on the target plate directly into the ion source (40 °C) in a glycerol matrix. The target was bombarded with xenon atoms having a maximum of 6 kV energy. The acceleration voltage of generated ions was 10 kV. The spectra were recorded at a scan range of 0-800 m/z. Calibration was based on solid CsI (cesium iodide, positive ion mode).
4.1.4. Antimicrobial assay The agar diffusion method was performed using a disc test and a spot test. The disc test was performed according to a procedure developed by Pulusani et al. (1979) with some modifications: 10 ml of the melted agar medium was seeded with 100 ìl of an 18 ± 2 h old broth culture of the test organism in a sterile petri dish. When the soft agar had hardened, an antibiotic test disc (diameter 6 mm, Schleicher & Schuell) was placed on the agar surface, and 22 ìl of the sample was spotted onto the disc. After incubation for 20 ± 2 h at the appropriate temperature for each organism tested, the diameter of the inhibition zone around the disc was measured. The spot test was done by spotting the liquid sample (3 ìl) directly onto the surface of the solidified, seeded agar medium, and the diameter of the inhibition zone was measured after incubation. Turbidometric assays were performed using a Bioscreen C automated turbidometer equipped with a Biolink software (Labsystems Co., Helsinki, Finland). The growth of indicator organisms in broth (300 ìl) containing antimicrobial compounds was studied in plates (100 wells). Each well was inoculated with 100 ìl broth culture (grown overnight) of the test organism diluted to 106 to 107 CFU ml-1. The optical density was measured automatically at 30 min-interval, using a wideband filter (405-600 nm), and the plates were shaken at 3 min-interval for 20 s. The growth curves were determined from the turbidity data.
4.2. EPSs produced by LAB (III, IV, V) 4.2.1. Bacterial strains and growth conditions The LAB strains examined for producing EPSs and their growth conditions are shown in Table 4. The source and methods of maintainance of these strains were the same as described above for the LAB strains examined for producing antimicrobial compounds in this study (4.1.1.)
Table 4. Lactic acid bacterial strains examined for producing exopolysaccharides, their growth media and incubation conditions used in this study Strain Growth medium Incubation condition Lactobacillus sp. fermentum G.1.2.1* Whey medium 37°C, 18 h helveticus Äki4 MRS 37°C, 45 h helveticus Lb161 Skim milk 37°C, 20 h helveticus K16* Skim milk 37°C, 24 h rhamnosus LC705* Skim milk 30°C, 24 h rhamnosus GG* Lactose-hydrolyzed milk 30°C, 20 h Lactococcus sp. lactis ssp. cremoris strains ARH 53, ARH 74, ARH 84, ARH 87, B30 Skim milk 25°C, 18-20 h lactis ssp. cremoris SEPH 11* Skim milk 25°C, 18-20 h Streptococcus thermophilus THS/41* Skim milk 37°C, 18 h * Unpublished. 4.2.2. Isolation of EPSs For the isolation of the EPS produced by Lb. helveticus Äki4 grown in MRS broth, bacterial cells were filtered from the medium, and the cell-free supernatant was concentrated 10- fold by lyophilization. The concentrate was fractionally precipitated with ethanol from 40 to 95% with intermediate centrifugation. The polysaccharide precipitated at 40% ethanol was washed, and dissolved in water. After filtration through a syringe filter (0.8 ì/0.2 ìl), it was freeze-dried. The crude polysaccharide (20 mg) was purified by anion-exchange chromatography with a column (25 x 1.5 cm) on Fractogel TSK DEAE-650(S) gel (Merck) using a Bio-Rad Econo system. The column was eluted at about 60 ml h-1 first with water for 80 min, and subsequently with 0.06 M NH4OAc adjusted to pH 5.5 with AcOH for 120 min. A fraction was collected every eight minutes with monitoring at 254 nm, and the presence of sugar was tested with a Molish reagent (Miller and Neuzil 1982).
For the isolation of the EPSs produced by other LAB strains grown in milk or whey medium (Table 4), proteins and cells were initially precipitated by addition of 4% (w/v) TCA (Merck) to the culture, and the mixture was stirred for 2 h. After centrifugation (35 min, 22 000 g, 4 °C), the supernatant was collected and filtered. Cold ethanol was then gradually added to the cell-free supernatant from one to two, and three volumes of the supernatant with intermediate centrifugation. The EPS precipitated was washed and dissolved in water. The aqueous solutions of EPS were filtered, and then extensively dialyzed against water overnight at 4 °C with two changes of water, and finally lyophilized. The purity of the EPS material was examined by gel filtration using a column (75 x 1.5 cm) of Bio-Gel P-30 polyacrylamide gel (exclusion limit 40 000 daltons, 100-200 mesh). The sample (1 mg) was loaded onto the column and eluted with 0.05 M NH4OAc with UV monitoring at 280 nm. To check the ionic nature of the EPSs, anionexchange chromatography of the EPS solutions (~1 mg mL-1) was performed using a column (25 x 1.5 cm) of weakly basic Fractogel TSK DEAE-650(S) gel. Elution was carried out at 1.1 mL min-1; first with water for 2 h, and subsequently with NH4OAc from 0.1 to 0.5 M using a linear increasing gradient.
4.2.3. Structural elucidation of EPSs GC analysis of alditol acetates was performed on a HP-5 fused silica column (0.20 mm x 25 m) using a temperature program of 180 °C for 1 min followed by 3 °C min-1 to 250 °C. Hydrogen was used as the carrier gas. The column was fitted to a Hewlett-Packard model 5890 series II gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA) equipped with a flame ionization detector. GLC-MS analysis was performed on a Hewlett-Packard model 5970 mass spectrometer equipped with an HP-5MS fused silica column (0.2 mm x 25 m). A temperature program of 170 °C for 3 min followed by 3 °C min-1 to 250 °C was used with helium as the carrier gas.
In sugar analysis, the EPS samples were hydrolyzed with 2 M TFA at 120 °C for 2 h. After reduction with sodium borohydride (NaBH4) and acetylation, the samples were analyzed by GC. The absolute configuration of the sugars present in the EPSs was determined essentially as devised by Leontein et al. (1978) but with (+)-2-butanol (Gerwig et al. 1978). Methylation analysis was performed according to Hakomori (1964) using sodium methylsulfinylmethanide and iodomethane in dimethyl sulfoxide. The methylated compounds were recovered by use of Sep-Pak C18 cartridges (Millipore) using the method of Waeghe et al. (1983). The purified methylated sample was then hydrolyzed (2 M TFA, 120 °C, 2 h), reduced, and acetylated. The partially methylated alditol acetates were analyzed by GLC-MS. NMR spectra of solutions in D2O were recorded at 65 °C and pD 5.5, using a Jeol GSX- 270, Jeol Alpha-400, or Varian Inova 600 or 800 MHz instrument. Chemical shifts are reported in ppm relative to sodium 3-trimethyl-(2,2,3,3-2H4)propanoate (äH 0.00) or acetone (äc 31.00) as internal references, or dioxan as an external reference. Data processing was performed using standard Jeol software, VNMR software, or Felix 2.3 (Biosym/MSI, San Diego, CA, USA). 1H,1H-COSY, relayed COSY, double-relayed COSY, TOCSY, 13C,1H-COSY, gHSQC (Wilker et al. 1993) and HMBC (Bax and Summers 1986) experiments were used to assign signals and performed according to standard pulse sequences. For inter-residue correlations, 2D NOESY experiments with mixing times of 300 and 400 ms (III), or 75 and 150 ms (IV), and HMBC experiments with 60 and 90 ms (III), or 45 and 90 ms (IV) delays for the evolution of long-range couplings were used.
4.2.4. Rheological measurements of the EPSs produced by Lc. lactis ssp. cremoris strains The viscosities of the dilute solutions of EPS at concentrations of 0.01 up to 0.1 g dL-1 were measured at 25 °C with an Ubbelohde capillary viscometer (536 13/Ic, SCHOTT35 GERÄTE, Hofheim, Germany), which allows the determination of the flow times with an accuracy of 0.03 s. The aqueous solutions of EPS with or without the addition of salt were prepared by dissolving a measured amount of EPS in 0.1 M sodium chloride (NaCl) solution or in deionized water. Sample dilution to the various required concentrations of EPS was done directly in the viscometer. After about 5 min for temperature equilibration, flow times were taken, and each flow time was reproduced six times. The reduced viscosity and intrinsic viscosity of the EPS solutions were calculated from the collected data.
The effect of temperature, pH and salts on the rheological behavior of the EPS solutions (1%, w /v) was studied with a Bohlin VOR rheometer (Bohlin Instruments Ltd, England) using concentric cylinders (C14) with a gap of 0.5 mm between the upper and lower geometries. The viscometry measurements were performed at 5, 25, 40 or 60 °C, with increasing shear rates up to 291 s-1 in 29 steps. The pH of the EPS solutions was adjusted with lactic acid to 4.0, 5.0 and 6.5. The EPS solutions containing salts were prepared by addition of EPS to 0.1 M NaCl or 0.1 M calcium chloride (CaCl2) solutions. The oscillation measurements were performed for the EPS (1%, w/v) in aqueous and salt solutions, and in skim milk at a frequency sweep from 0.01 to 15 Hz in 19 steps. For every temperature-dependent measurement a thermal equilibration time of about 60 min was used.
5. RESULTS AND DISCUSSION 5.1. Antimicrobial compounds produced by LAB (I, II, unpublished results) 5.1.1. Separation, purification and identification of antimicrobial compounds After ethanol precipitation of the cell-free cultures of the LAB strains (Table 3), the obtained precipitates and the final supernatants were tested for antimicrobial activity. The supernatants containing antimicrobial activity were subjected to chromatographic separation and purification. The active fractions appeared in a relatively narrow part of the chromatogram on gel-filtration on Bio-Gel P-2 of the supernatants (Fig. 1/I). Anion exchange chromatography of these active fractions (Fig. 2/I) resulted in two different ranges of active fractions (52-54 and 58- 63), fractions 58-63 containing lactic acid. Further purification by RP-HPLC of the fractions 52- 54 gave rise to one major peak at retention time 4.96 min and two small peaks at 3.65 and 5.78 min. The absorption maximum of the antimicrobial compound was at 215 nm (Fig. 3/I). Fractions of these peaks were collected and tested for antimicrobial activity. Only the fraction of the peak at 4.96 min was found to contain antimicrobial activivity. Identification of this active fraction by both NMR (1H and 13C) and MS (EI and FAB) spectra indicated that the antimicrobial compound was 2-pyrrolidone-5-carboxylic acid (PCA), also known as pyroglutamic acid. Among the twenty LAB strains (Table 3) examined, thirteen Lactobacillus and five Pediococcus strains were found to produce PCA under the growth conditions used in this study (Table 5). Four Lactobacillus strains produced, in addition to PCA, also HMM antimicrobial compounds, as indicated by the presence of antimicrobial activity in the precipitates obtained from ethanol precipitation. Since these HMM compounds exhibited a rather narrow range of activity (Table 5) they were not subjected to further studies. On the basis of the results of this study, it seems that many LAB strains, particularly Lactobacillus strains, are able to produce PCA.
Since LAB produce relatively large amounts of lactic acid, being antimicrobially active, it is important to remove the lactic acid in order to find other antimicrobial compounds, especially those of low molecular mass. In the separation and purification procedures developed in this study, both PCA and lactic acid were present in the culture supernatant obtained from ethanol precipitation, and they also appeared in almost the same range of fractions after gel filtration. However, complete separation of lactic acid was achieved by anion exchange chromatography based on a gel matrix (Fractogel TSK) suitable for separation of biomolecules. Previously, size exclusion HPLC was used to separate lactic acid from LMM antimicrobials, but all the fractions obtained were found to contain antimicrobial activity because the mobile phase (sodium phosphate) used was antimicrobially active (Lortie et al. 1993). Niku-Paavola et al. (1999) reported the separation of lactic acid by gel filtration on Sephadex G-10 with water as an eluent and found several LMM antimicrobial compounds produced by Lb. plantarum. Although there were some reports on the separation and purification of LMM antimicrobial compounds produced by LAB (Pulusani et al. 1979, Reddy and Ranganathan 1983, Mehta et al. 1984), the techniques for seperation of lactic acid appeared not to be clearly demonstrated. In addition, there were reports (Nielsen et al. 1990) on the use of a neutralization technique to eliminate the antimicrobial effect of lactic acid, but lactic acid could not be separated with this technique, and the activity of acidic antimicrobials might be suppressed due to neutralization. PCA is a natural constituent of foods of plant origin, including vegetables and fruits (Airaudo et al. 1987), and fermented soybean and cereal products (Masaaki et al. 1992, Syuhei et al. 1994). Among LAB strains, only S. bovis has previously been shown to produce PCA by conversion of glutamine (Chen and Russel 1989). PCA can also be synthesized by heating glutamic acid using a dehydration process (Mijin et al. 1989). It has been reported that PCA is able to increase cerebral blood flow and decrease the resistance of brain vessels, which result in enhanced brain metabolism, i.e. increased glucose uptake and utilization by cerebral tissues and decreased brain lactate dehydrogenase activity (Mirizoian et al. 1994). Other biological functions of PCA are related to its presence as an amino-terminal residue in many biologically significant peptides and proteins, e.g., eisenine, LH-RH (luteinizing hormone releasing hormone), and TRH (thyrotropin releasing hormone) (Pattabhi and Venkatesan 1974, Abraham and Podell 1981, Paul et al. 1990).
Table 5. Lactic acid bacterial strains producing antimicrobial compounds and the sensitive strains for the HMM compounds (Table 1 in paper II and unpublished results) Producing strain Antimicrobial compound Sensitive strains for the HMM compounds* Lactobacillus sp. acidophilus "NCFB" Lb1748 PCA HMM compounds* Lb. delbrueckii ssp. bulgaricus KKNO 293 Lb. helveticus 632 KKNO 1129 L. lactis ssp. lactis Lb430 KKNO 1512 L. lactis ssp. lactis KKNO 1189 casei C PCA casei ssp. casei LC-10 PCA casei LC1/6-1 PCA casei SHIROTA PCA delbrueckii ssp. delbrueckii strain 13S PCA HMM compounds* Lb. delbrueckii ssp. bulgaricus KKNO 293 helveticus Äki4 PCA HMM compounds* Lb. delbrueckii ssp. bulgaricus KKNO 293 lactis KKNO 1134 Lb78 PCA lactis ssp. bulgaricus KKNO 312 Lb389 PCA paracasei ssp. paracasei Lb1931 PCA reuteri DSM20016 PCA HMM compounds* Lc. lactis ssp. lactis Lb430 KKNO 1512 rhamnosus GG PCA |
