Microbial physiology, fermentation kinetics, and process engineering of heteropolysaccharide production by lactic acid bacteria

International Dairy Journal 11 (2001) 747–757

Microbial physiology, fermentation kinetics, and process engineering of heteropolysaccharide production by lactic acid bacteria Bart Degeest, Frederik Vaningelgem, Luc De Vuyst* Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing, Vrije Universiteit Brussel, IMDO, Pleinlaan 2, B-1050 Brussels, Belgium

Abstract Many species of lactic acid bacteria (LAB) produce extracellular heterotype polysaccharides, the so-called heteropolysaccharides (HePS). Biosynthesis and secretion of the HePS from the LAB occur during different growth phases, and both the amount and type of the polymer is influenced by growth conditions. The total yield of exopolysaccharides produced by the LAB depends on the composition of the medium (carbon and nitrogen sources, growth factors, etc.) and the conditions in which the strains grow, i.e. temperature, pH, oxygen tension, and incubation time. It is never higher than 1.5 g of polymer dry mass per litre of fermentation medium. Whereas mesophilic strains produce maximal amounts of HePS under conditions not optimal for growth, the HePS production from thermophilic LAB strains is growth-associated, i.e. maximum production during growth and under conditions optimal for growth. The HePS degradation often takes place upon prolonged incubation of the HePS-producing LAB strains due to glycohydrolase activity. Primary, secondary, and tertiary modelling unravel the functionality of the HePS-producing LAB strains in a food environment. Finally, appropriate process engineering can lead to an industrial breakthrough of the HePS production and applications: a high and stable high-molecular-mass HePS production by appropriate feeding strategies through fed-batch cultivation on the one hand, and the application of a two-step fermentation process in yoghurt manufacture on the other hand. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Exopolysaccharides; Heteropolysaccharides; Lactic acid bacteria

1. Introduction Many species of lactic acid bacteria (LAB) produce extracellular polysaccharides (EPS) (Cerning, 1990, 1995; Cerning & Marshall, 1999; De Vuyst & Degeest, 1999; Ricciardi & Clementi, 2000). EPS is a general term that refers to two types of secreted polysaccharides (Sutherland, 1972). The first type of EPS is attached to the cell wall as a capsule (capsular polysaccharides or CPS), while the other is produced as loose unattached material (slime EPS, further referred to as EPS). Some strains produce both types of EPS, whereas others produce only one type. Strains of the LAB that are able to produce EPS in large enough quantities are an interesting alternative source of polymers for use as food additives. In addition, those microorganisms can be *Corresponding author. Tel: +32-2-629-32-45; fax: +32-2-629-2720. E-mail address: [email protected] (L. De Vuyst).

used for the in situ production of EPS, in particular in fermented dairy products (yoghurt and cheese) to improve the rheology, texture and body, and mouthfeel of these food products. EPS from the LAB can be subdivided into two groups, namely homopolysaccharides (HoPS) and heteropolysaccharides (HePS). Whereas the HoPS are composed of one type of constituting monosaccharides, the HePS are composed of a backbone of repeated subunits (Monsan et al., 2001; De Vuyst, De Vin, Vaningelgem, & Degeest, 2001). Many different types of HePS are secreted by several LAB strains with respect to sugar composition and molecular mass, the latter varying from 1.0
104 to 6.0
106 (Cerning, 1995; De Vuyst & Degeest, 1999). However, contradictory results have been reported regarding the influence of physical and chemical factors on the HePS production by the LAB (De Vuyst & Degeest, 1999). This review focuses on the physiology, fermentation kinetics, and process engineering of the HePS from the LAB.

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2. Ropy and slimy character of exopolysaccharideproducing lactic acid bacterium strains Biosynthesis and secretion of the HePS from the LAB occur during different growth phases, and both the amount and type of the polymer is influenced by growth conditions (De Vuyst & Degeest, 1999). Moreover, several strains of the LAB have been shown to express at least two distinct phenotypic forms of the HePS, ropy and mucoid (Dierksen, Ebel, Marks, Sandine, & Trempy, 1995; Dierksen, Sandine, & Trempy, 1997; Knoshaug, Ahlgren, & Trempy, 2000). Whereas ropy HePS is defined phenotypically by viscous ropes longer than 5 mm, originating from the colony when the colony is touched, mucoid HePS imparts a slimy appearance to the colony but does not produce viscous ropes. Finally, capsule production occurs among non-ropy and ropy LAB strains. For instance, Hassan, Frank, and Shalabi (2001) observed that a non-ropy strain of Streptococcus thermophilus produced 5-mm diameter capsules when grown in milk and only 2.0–2.5-mm diameter capsules in Elliker broth (Hassan, Frank, Farmer, Schmidt, & Shalabi, 1995). They observed that differences in the sugar content of milk and Elliker broth, led to the formation of different size capsules (Hassan et al., 2001).

3. Media used for heteropolysaccharide production by lactic acid bacteria Since no HePS production was initially observed in MRS or synthetic media, milk was often the medium studied (Cerning, Bouillanne, Desmazeaud, & Landon, 1986, 1988; Cerning, Bouillanne, Landon, & Desmazeaud, 1990, 1992; Garcia-Garibay & Marshall, 1991; De Vuyst, Vanderveken, Van de Ven, & Degeest, 1998). Also, whey and whey-based media have been used (Ariga et al., 1992; Gassem, Schmidt, & Frank, 1997; Knoshaug et al., 2000). However, only recently have semi-synthetic and synthetic media been investigated (Cerning et al., 1994; Grobben, Sikkema, Smith, & De Bont, 1995; Grobben et al., 1998; van den Berg et al., 1995; Kimmel & Roberts, 1998; Dupont, Roy, & Lapointe, 2000; Petry, Furlan, Crepeau, Cerning, & Desmazeaud, 2000; Torino, Sesma, & Font de Valdez, 2000b). A chemically defined medium containing a carbohydrate source, amino acids, vitamins, nucleic acid bases, and mineral salts is indeed more suitable to investigate the influence of nutrients on growth, metabolic pathways, and biosynthesis of the HePS in the LAB. It allows the quantitative and qualitative production of the HePS and the investigation of the exact composition of the HePS produced. Media containing complex nutrients like beef extract, peptone, and yeast extract are not suitable because of interference of these

compounds with the monomer and structure analysis of the HePS (Kimmel & Roberts, 1998; Torino et al., 2000b; Degeest, Vaningelgem, Laws, & De Vuyst, 2001b). Recently, Petry et al. (2000) developed a chemically defined medium that may be useful as an alternative to milk in studies of the HePS production, because as much HePS was found in the former medium under pH-controlled conditions as compared to the latter.

4. Exopolysaccharide yields produced by lactic acid bacteria The total yield of EPS produced by the LAB depends on the composition of the medium (carbon and nitrogen sources, growth factors, etc.) and the conditions in which the strains grow, i.e. temperature, pH, oxygen tension, and incubation time (cf. infra). The yield of intracellularly synthesized HePS by different LAB strains varies roughly from 0.045 to 0.350 g L1 when the bacteria are grown under non-optimized culture conditions. Optimal culture conditions result in HePS yields from 0.150 to 0.600 g L1, depending on the strain (Cerning, 1990, 1995; Cerning & Marshall, 1999; Ricciardi & Clementi, 2000). When a ropy strain of S. thermophilus is grown in association with a non-ropy strain of Lactobacillus delbrueckii subsp. bulgaricus in milk, the HePS production can reach quantities of almost 0.800 g L1 (Cerning et al., 1988, 1990). An optimal carbon/nitrogen ratio in both milk and MRS media gives yields of 1.5 g L1 of HePS with S. thermophilus LY03 (De Vuyst et al., 1998; Degeest & De Vuyst, 1999, 2000). With Lactobacillus sakei 0-1 and Lactobacillus rhamnosus 9595M, HePS yields of approximately 1.4 and 1.3 g L1, respectively, are achieved (van den Berg et al., 1995; Dupont et al., 2000; Degeest, Janssens & De Vuyst, 2001a). These values are however not comparable with the high yields obtained with the dextran-producing LAB (van Geel-Schutten, Flesch, ten Brink, Smith, & Dijkhuizen, 1998) and Gram-negative EPS producers such as Xanthomonas campestris (De Vuyst, Van Loo, & Vandamme, 1987).

5. Nutrients enhancing growth and heteropolysaccharide production of lactic acid bacteria 5.1. Heteropolysaccharide production in milk media Enhanced HePS production and growth were initially obtained when (hydrolyzed) casein was added to skim milk cultures of Lb. delbrueckii subsp. bulgaricus (Cerning et al., 1990; Garcia-Garibay & Marshall, 1991). According to early investigations, neither growth nor HePS production was specifically linked to the

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presence of casein or whey proteins in the growth medium of the LAB. However, Cerning et al. (1990) found that casein stimulates the HePS production, but not growth of Lb. delbrueckii subsp. bulgaricus. On the other hand, addition of hydrolyzed casein to MRS does not increase specific polymer production by Lb. delbrueckii subsp. bulgaricus (Garcia-Garibay & Marshall, 1991), although later work by Kimmel, Roberts, and Ziegler (1998) suggests that its supplementation enhances the HePS production. It has further been reported that Lb. delbrueckii subsp. bulgaricus is able to produce the same amount of HePS in milk and milk ultrafiltrate, but that S. thermophilus is not (Cerning et al., 1990). On the other hand, supplementation of milk and milk ultrafiltrate with glucose or sucrose stimulates the HePS production by Lactobacillus casei and even modifies the monosaccharide composition of the HePS with glucose becoming dominant. In the latter case, rhamnose as HePS-constituting residue is no longer present as compared to growth and HePS production in a synthetic medium (Cerning et al., 1992, 1994; Kojic et al., 1992). Lb. delbrueckii subsp. bulgaricus NCFB 2772 produces considerably larger amounts of the HePS when grown on glucose or lactose than when grown on fructose to equal cell densities, and the HePS have a different monomer sugar composition (cf. infra). Compared with growth on the other carbohydrate sources, growth on mannose leads towards much lower levels of growth and HePS production (Grobben et al., 1995; Grobben, Smith, Sikkema, & De Bont, 1996). Mannose or a combination of glucose and fructose are the most efficient carbon sources for the HePS production by Lb. rhamnosus C83 (Gamar, Blondeau, & Simonet, 1997). For Lactococcus lactis, a higher HePS production and a better cell growth is observed for growth on glucose compared to fructose, although the transcription level of the eps gene cluster is independent of the carbohydrate source (Looijesteijn, Boels, Kleerebezem, & Hugenholtz, 1999). Also, Petry et al. (2000) found that the carbohydrate source (glucose or lactose) influences the HePS production by Lb. delbrueckii subsp. bulgaricus CNRZ 1187 in a chemically defined medium. In addition, not only the nature of the carbon source and sometimes the combination of monosaccharides, but also their concentration can have a stimulating effect on the HePS biosynthesis (Cerning et al., 1992, 1994; Gamar et al., 1997; Degeest & De Vuyst, 2000). As an example, when Lb. rhamnosus C83 is grown in a chemically defined medium on 4% mannose or 2% glucose and fructose (ratio 1 : 1), it was found that the HePS production increases by three or four times whereas the final biomass concentrations are identical (Gamar et al., 1997). Similarly, when S. thermophilus LY03 and S. thermophilus Sfi20 are grown in a medium with both glucose (2.5% and 5.0%, respectively) and lactose (5.0% and 2.5%, respectively),

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the HePS production is enhanced as compared to growth in media with glucose (7.5%) or lactose (7.5%) as the sole carbohydrate sources (Degeest & De Vuyst, 2000; Degeest et al., 2001b). Finally, high carbohydrate content and other milk components such as whey protein stimulate capsule production of both ropy and non-ropy strains of S. thermophilus and Lb. delbrueckii subsp. bulgaricus in milk (Hassan et al., 2001). 5.2. Carbon, nitrogen, phosphate, and other nutrients limitation during heteropolysaccharide production For some HePS-producing bacteria, such as Xanthomonas, Pseudomonas, and Rhizobium spp., nitrogen limitation results in increased HePS production (Sutherland, 1990). This seems not to be the case for the LAB strains (De Vuyst et al., 1998; Kimmel et al., 1998; Sebastiani & Zelger, 1998). Moreover, it has been shown that an optimal balance between the carbon and nitrogen source is absolutely necessary to achieve high HePS yields (Degeest & De Vuyst, 1999, 2000; De Vuyst et al., 1998). This may be explained by the fact that the LAB are dependent on the nitrogen source for cell synthesis, whereas the carbon source is mainly utilized for energy generation. Marshall, Cowie, and Moreton (1995) showed that L. lactis subsp. cremoris LC 330 produces two HePS simultaneously: a neutral HePS with a molecular mass higher than 1.0
106 and a charged phosphopolysaccharide with a molecular mass of approximately 1.0
104. In contrast to the low-molecular-mass HePS, the production of the high-molecular-mass HePS is positively influenced by nitrogen limitation. Looijesteijn, van Casteren, Tuinier, Doeswijk-Voragen, and Hugenholtz (2000) showed that the type of substrate limitation has a remarkable influence on the molecular mass of the HePS produced by L. lactis subsp. cremoris NIZO B40. Under glucose/energy limitation, the molecular mass is much smaller than under leucine or phosphate limitation. Degeest and De Vuyst (1999) observed a ‘shift’ from high-molecular-mass to low-molecular-mass HePS production by S. thermophilus LY03 with increasing initial concentrations of the complex nitrogen source. However, both the HePS forms degraded upon prolonged fermentation (Degeest, Mozzi, Vaningelgem, & De Vuyst, unpublished results). In contrast, when both the carbon/nitrogen ratio and lactose pressure were kept high through fed-batch cultivation, a high high-molecular-mass HePS production and a stable high-molecular-mass HePS concentration, respectively, were observed (Vaningelgem, Degeest, & De Vuyst, unpublished results). Further, the possibility exists that other medium components influence the HePS production as well. For instance, mineral requirements would affect the HePS production (Gamar et al., 1997; Mozzi, Savoy de Giori,

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Oliver, & Font de Valdez, 1995b; Torino et al., 2000b). Also, vitamins seem to play an important role (Grobben et al., 1998). In contrast to the omission of single or multiple amino acids, it was demonstrated that the omission of multiple vitamins affects the production of the HePS relative to cell growth. However, it only affects the total amount of the HePS produced and not the ratio of the high-molecular-mass fraction and the lowmolecular-mass fraction of the HePS from Lb. delbrueckii subsp. bulgaricus NCFB 2772 as is found when fructose instead of glucose is used as the carbohydrate source (cf. infra). The requirement observed for vitamins could, however, depend on the presence of lipids in the medium, e.g. oleate. Petry et al. (2000) found that orotic acid influences the HePS production by Lb. delbrueckii subsp. bulgaricus in a chemically defined medium. Finally, the yields of the phosphorylated HePS from L. lactis NIZO B40 as well as the unphosphorylated HePS from L. lactis NIZO B891 are reduced with about 40% under conditions of phosphate limitation (Looijesteijn et al., 2000).

6. Influence of the carbon and nitrogen source on heteropolysaccharide composition The influence of the carbon source on the nature and distribution of sugars in the HePS produced by the LAB has long been debated, particularly because minor differences in the composition of the polymer were often found due to analytical artefacts (De Vuyst & Degeest, 1999). For instance, Petit, Grill, Maazouzi, and Marczak (1991), showed that polysaccharide production increases and that carbohydrate and uronic acid distribution in polysaccharides alters in favour of galactose with decreasing lactose feed rate in fed-batch cultivation with a galactose-fermenting S. thermophilus strain. Unfortunately, no structure determinations were carried out. Grobben et al. (1996) showed that in continuous culture, when the Lb. delbrueckii subsp. bulgaricus NCFB 2772 strain is grown on lactose, the amount and sugar composition of the HePS produced is comparable with the values for glucose-grown cultures, i.e. galactose, glucose, and rhamnose in a ratio of 6.8 : 1.0 : 0.7. When grown with fructose as the carbohydrate source, the amount of the HePS produced is substantially lower and the HePS are different in composition, being composed of galactose and glucose in the ratio 2.5 : 1.0. No rhamnose residues were detected in these HePS. The authors concluded that Lb. delbrueckii subsp. bulgaricus NCFB 2772 produces two HePS fractions with relative molecular masses of 1.7
106 and 4.0
104. Later, they reported on a different monomer composition, and found that the production of the high-molecular-mass fractions (galac-

tose, glucose, and rhamnose in the molar ratio of 5.0 : 1.0 : 1.0) appears to be dependent on the carbohydrate source (glucose versus fructose), whereas the lowmolecular-mass fractions (galactose, glucose, rhamnose in the molar ratio of approximately 11.0 : 1.0 : 0.4) are produced more continuously (Grobben et al., 1997). The two Lb. delbrueckii subsp. bulgaricus strains studied by Petry et al. (2000) did not show the same behaviour with two different carbohydrate sources; for Lb. delbrueckii subsp. bulgaricus CNRZ 416, the proportion of the monosaccharide constituents varies as a function of the carbohydrate source, while for Lb. delbrueckii subsp. bulgaricus CNRZ 1187, it does not. Similarly, no differences in sugar composition in the HePS produced by other different LAB strains was found when growth on different carbohydrate sources is compared (Manca de Nadra, Strasser de Saad, Pesce de Ruiz Holgado, & Oliver, 1985; van den Berg et al., 1995; Gamar-Nourani, Blondeau, & Simonet, 1998; Degeest & De Vuyst, 1999, 2000; Looijesteijn et al., 2000). These differences of the influence of the carbohydrate source(s) on the HePS monomer composition may highlight variations in the HePS regulation and biosynthesis in different strains. Hence, additional information on the effects of varying growth conditions on the size and composition of the HePS produced and, consequently, the functional and rheological properties of the HePS from the LAB would be of significant benefit to the food industry.

7. Influence of temperature, pH, and oxygen tension on heteropolysaccharide production by lactic acid bacteria Optimal conditions of temperature, pH, oxygen tension, agitation speed, and incubation time, result in improved HePS yields (Gancel & Novel, 1994a, b; Mozzi, Savoy de Giori, Oliver, & Font de Valdez, 1994, 1996a, b; Mozzi, Oliver, Savoy de Giori, & Font de Valdez, 1995a; Mozzi et al., 1995b; van den Berg et al., 1995; Gamar et al., 1997; Gassem et al., 1997; De Vuyst et al., 1998; Gamar-Nourani et al., 1998; Looijesteijn & Hugenholtz, 1999; Petry et al., 2000; Torino, Mozzi, Sesma, & Font de Valdez, 2000a). Several studies show that low temperatures markedly induce slime production (Forse! n, Raunio, & Myllymaa, 1973; Kontusaari & Forse! n, 1988; Cerning et al., 1992; Kojic et al., 1992; Gancel & Novel, 1994a; Marshall et al., 1995; van den Berg et al., 1995; Mozzi et al., 1995a, 1996a; Gamar et al., 1997; Gassem et al., 1997; Breedveld, Bonting, & Dijkhuizen, 1998; Looijesteijn & Hugenholtz, 1999). Based on information for the HePS production from Gram-negative bacteria, this effect has been explained as follows. Slowly growing cells exhibit much slower cell wall polymers biosynthesis, making more isoprenoid lipid carrier precursor molecules available for the HePS biosynthesis (Sutherland, 1972).

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Gamar-Nourani et al. (1998) demonstrated that a temperature shift (from 371C to 251C) at the beginning of the exponential growth phase enhances the HePS production by Lb. rhamnosus C83. However, several investigators find higher HePS production by the LAB strains at higher cultivation temperatures (GarciaGaribay & Marshall, 1991; Grobben et al., 1995; Mozzi et al., 1995a; De Vuyst et al., 1998) and under conditions optimal for growth, for instance with respect to pH (Mozzi et al., 1994, 1996a; De Vuyst et al., 1998; Grobben et al., 1998; Gamar-Nourani et al., 1998; Petry et al., 2000) and oxygen tension (De Vuyst et al., 1998; Grobben et al., 1998; Petry et al., 2000). Aeration is not required, since higher HePS yields are obtained with a lower oxygen tension (De Vuyst et al., 1998; GamarNourani et al., 1998; Looijesteijn & Hugenholtz, 1999) as well as anaerobically (van den Berg et al., 1995; De Vuyst et al., 1998; Degeest, Van de Ven, & De Vuyst, 1999; Petry et al., 2000). Aeration may, however, become a problem under the conditions that the HePS are produced using engineered strains with improved HePS production capacity, for instance, the LAB strains capable of catabolizing galactose and producing additional ATP through the production of acetate via acetate kinase. Agitation does not influence growth nor HePS production markedly; it might, however, affect viscosity probably breaking some molecular associations among the HePS, certain medium components, and the bacterial surface (Torino et al., 2000a). Optimal pH conditions for production of the HePS are often close to pH 6.0 (Mozzi et al., 1994, 1996a; van den Berg et al., 1995; Gassem et al., 1997; De Vuyst et al., 1998; Grobben et al., 1998; Looijesteijn & Hugenholtz, 1999). Van den Berg et al. (1995) postulated that conversion of sugar to HePS is more efficient at pH 5.8, but sugar is more efficiently converted to biomass at pH 6.2. Maintenance of a higher pH will result in increased HePS production by increasing the time the culture is in the exponentional growth phase (Gassem et al., 1997; Gamar-Nourani et al., 1998). Higher pH also results in a longer stationary phase, which would decrease peptidoglycan and teichoic acid syntheses and could result in increased HePS production. It has further been shown that the HePS production under growth conditions with continuously controlled pH is significantly higher than in acidifying batch cultures; moreover, it seems that the effect of pH adjustment is greater than that of supplementation with nutrients (Grobben et al., 1995, 1998; Mozzi et al., 1996a; Gassem et al., 1997; De Vuyst et al., 1998). The pH effect could be an important limiting factor when considering industrial exploitation of the HePS-producing LAB strains in fermented milks production since the latter takes place under free pH conditions. Alternatively, some authors do not find significant differences in the quantity of the HePS produced under controlled pH conditions as

751

compared to acidifying conditions (Gamar-Nourani et al., 1998; Dupont et al., 2000). Acidifying conditions may further have a positive influence on the stability of the HePS produced (Degeest, Mozzi, Vaningelgem, & De Vuyst, unpublished results).

8. Kinetics of heteropolysaccharide production by lactic acid bacteria 8.1. Primary versus secondary metabolite kinetics Whereas mesophilic strains seem to produce maximal amounts of HePS under conditions not optimal for growth, for instance low temperatures, the HePS production from thermophilic LAB strains appears to be growth-associated, i.e. maximal production during growth and under conditions optimal for growth (De Vuyst et al., 1998; Fig. 1). In case of growth-associated production, the HePS biosynthesis generally starts almost simultaneously with growth, shows a maximum rate when the culture is in its exponential growth phase, and reaches a maximum towards the end of the active growth, indicating primary metabolite kinetics (Manca de Nadra et al., 1985; Grobben et al., 1995; van den Berg et al., 1995; Gamar et al., 1997; De Vuyst et al., 1998; Kimmel et al., 1998; Knoshaug et al., 2000). In L. lactis subsp. cremoris Ropy352, expressing two phenotypically distinct HePS, optimal growth conditions parallel optimal production of the ropy HePS, whereas poor growth conditions parallel optimal production of the mucoid HePS (Knoshaug et al., 2000). Marshall et al. (1995) and Pham, Dupont, Roy, Lapointe, and Cerning (2000) indicated that the onset of the HePS biosynthesis from a strain of L. lactis subsp. cremoris and Lb. rhamnosus, respectively, is observed towards the end of the exponential growth phase. Other investigators observed continued HePS production beyond or only in the stationary phase, and hence consider the HePS as secondary metabolites (Manca de Nadra et al., 1985; Kojic et al., 1992; Gancel & Novel, 1994a; Bouzar, Cerning, & Desmazeaud, 1996; Grobben et al., 1998; Looijesteijn & Hugenholtz, 1999; Petry et al., 2000). A possible interpretation is that isoprenoid phosphate carriers are primarily needed for cell wall synthesis during growth. Upon cessation of growth, there is a greater availability of this molecule for the HePS biosynthesis (Petry et al., 2000). Gassem, Schmidt, and Frank (1995) also found that there is no association between growth rate or acid production and the HePS production in different media by the LAB strains. However, whereas the concentrations of peptidoglycan precursors are indeed shown to reach maximum values during exponential growth but are still present at high levels in the stationary phase in case of L. lactis subsp. cremoris NIZO B40, the concentrations of the sugar

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Fig. 1. Growth-associated biosynthesis of the HePS by S. thermophilus LY03 in a complex medium (MRS with 3.0% (m/v) peptone, 1.2% (m/v) yeast extract and 7.5% (m/v) lactose) at 421C and a controlled pH of 6.2. Cell growth (’, g CDM L1), substrate consumption (~, g lactose L1), excretion of galactose (n, g galactose L1), and the production of functional metabolites (m, g lactic acid L1;K, ½EPS in mg PDM L1).

nucleotides UDP-glucose and UDP-galactose, precursor molecules of the HePS building blocks, reach maximum values during exponential growth and are absent in the stationary phase of this strain (Ramos, Boels, de Vos, & Santos, 2001). Finally, with L. lactis subsp. cremoris NIZO B40, most of the HePS are produced during the exponential growth phase when the cells are grown on glucose, while during growth on fructose, about 60% of the HePS are produced in the stationary phase (Looijesteijn et al., 1999). The HePS production by Lb. rhamnosus R continued beyond the decline growth phase in the case of glucose-grown cells, whereas no further HePS production is found after growth has ceased in the medium supplemented with lactose (Pham et al., 2000). Therefore, as stated by Pham et al. (2000), the LAB HePS should be considered as minor products diverted away from glycolysis rather than as secondary metabolites. However, conditions like temperature, pH, growth rate, and nutrients that favour either growth or HePS production might allow the uncoupling of growth and HePS production in mesophiles (Looijesteijn & Hugenholtz, 1999). Whether this can be applied for thermophilic LAB strains needs further studies. Anyway, because of this growth-associated HePS production in thermophilic LAB strains and because of the limited number of catabolic pathways which provide energy in the LAB (substrate level phosphorylation and secondary metabolic energy generation), cell synthesis is limited, and so is the energy-demanding HePS biosynthesis. Consequently, improving the HePS production must be sought in enhancing biomass formation. Also with mesophilic LAB, the HePS production may be further enhanced on varying the environmental factors, once enough cells have been formed in the trophophase.

8.2. Heteropolysaccharide composition during fermentation Whereas Bouzar et al. (1996) report that the sugar composition of the HePS from Lb. delbrueckii subsp. bulgaricus CNRZ 1187 changes during the fermentation cycle, De Vuyst et al. (1998) found that the HePS composition of S. thermophilus LY03 remains constant during the whole batch fermentation process. In view of the biosynthesis mechanism involved, namely polymerization of building blocks, the latter will be affected by the activities of different sugar activation and interconversion enzymes, which in turn will depend on the available substrates or specific stimuli from the environment. Hence, a variable HePS composition during fermentation could be expected when varying substrates are fed to the microorganism. Alternatively, in case of specific stimuli from the environment, different enzymes involved in the HePS biosynthesis might be switched on or off, independent of the substrate used, and, as a result, the HePS composition might be affected accordingly. For instance, the phosphorus/carbohydrate and glucose/galactose ratio of the Lb. helveticus ATCC 15807 HePS, when the strain is grown in milk, is dependent on the fermentation time as well as on the culture conditions (Torino et al., 2000a). Petry et al. (2000) showed that relative monosaccharide ratios in the HePS of Lb. delbrueckii subsp. bulgaricus grown in a chemically defined medium are affected by the pH. The possibility that varying process conditions might influence the HePS composition during fermentation is under investigation currently (Vaningelgem & De Vuyst, unpublished results).

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8.3. Heteropolysaccharides breakdown upon prolonged fermentation The HePS degradation often takes place upon prolonged incubation of the HePS-producing LAB strains (Mozzi et al., 1996b; Gassem et al., 1997; De Vuyst et al., 1998; Petry et al., 2000; Pham et al., 2000). This may be due to glycohydrolase activity (Macura & Townsley, 1984; Cerning et al., 1988, 1990, 1992; Gancel & Novel, 1994b; Pham et al., 2000) that is rather stable towards temperature and pH (Degeest, Mozzi, Vaningelgem, & De Vuyst, unpublished results). The presence of these glycohydrolases has only been demonstrated for the HePS production by Lb. rhamnosus R (Pham et al., 2000). Indeed, a marked reduction in the HePS yield upon prolonged fermentation seems to be dependent on the strain and culture conditions used (temperature, pH, carbohydrate source, etc.) (Gancel & Novel, 1994b; Mozzi et al., 1994, 1995a; Gassem et al., 1997; De Vuyst et al., 1998; Pham et al., 2000; Degeest, Mozzi, Vaningelgem, & De Vuyst, unpublished results). Harvesting the HePS at the appropriate time and under the appropriate conditions of pH and temperature during isolation may avoid this problem. 8.4. Modelling of heteropolysaccharide production by lactic acid bacteria Primary modelling of cell growth, sugar consumption, and the production of lactic acid and EPS under a set of environmental conditions, is based on basic differential equations (Leroy, Degeest, & De Vuyst, 2001). In general, cell growth of the LAB may be modelled as d½X=dt ¼ mmax g½X;

ð1Þ

where ½X is the biomass concentration (in g CDM L1), mmax is the maximum specific growth rate (in h1), and g is an inhibitory function due to nutrient limitation, endproduct inhibition, food structure, microbial interactions, etc. In the case of the logistic equation, g is simplified as ð1  ½X=½Xmax Þ; with ½Xmax the maximum cell concentration (g CDM L1) for a given set of conditions (Lejeune, Callewaert, Crabbe! , & De Vuyst, 1998; Degeest & De Vuyst, 1999). Depletion of fermentable sugar (½S in g L1) may be modelled as follows (Pirt, 1965): d½S=dt ¼ 1=YX=S d½X=dt  mS ½X;

ð2Þ

where YX=S is the cell yield coefficient (g CDM (g fermentable sugar)1), and mS is the cell maintenance coefficient (g fermentable sugar (g CDM)1 h1). This equation states that the energy obtained from sugar consumption is simultaneously used for cell construction and maintenance processes.

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The following equations describe functional properties of the LAB, including the production of lactic acid (½L in g lactic acid L1) and EPS (½EPS in mg PDM L1) (Degeest & De Vuyst, 1999): d½L=dt ¼ YL=S d½S=dt;

ð3Þ

d½EPS=dt ( if ½Xo½X 0  ; kf d½X=dt ¼ kd ð½EPS  ½EPSr Þ if ½X4½X 0  or ½S ¼ 0: ð4Þ Biokinetic parameters are the yield coefficient for the production of lactic acid YL=S (g lactic acid (g fermentable sugar)1), the specific EPS formation kf (mg PDM (g CDM)1), the critical biomass concentration ½X 0  (g CDM L1) at which EPS production ceases, the EPS degradation rate kd (h1), and the residual concentration of EPS (½EPSr in mg PDM L1). The Eqs. (1)–(4) have been successfully applied for the primary modelling of the experimental data obtained with the HePS-producing S. thermophilus LY03 strain (Degeest & De Vuyst, 1999). Primary modelling indicates that the production of EPS occurs in a growth-associated manner; upon prolonged fermentation, EPS are degraded (cf. infra). Conditions that provide high cell density are hence likely to favour a high production of EPS. However, not only the amount of producing cells will determine production characteristics, the amount of metabolite produced per cell, i.e. the specific metabolite production, has to be taken into account as well. The specific production and the degradation of EPS are dependent on the environment (cf. infra). Secondary modelling consists of expressing the biokinetic parameters of the Eqs. (1)–(4) (mmax ; YX=S ; mS ; YL=S ; kf ; kd ; ½X 0 ; ½EPSr ) as a function of a single environmental factor (e.g. temperature). Establishing mathematical expressions of biokinetic parameters such as the specific EPS production kf and the EPS degradation rate kd as a function of a combined set of environmental factors may yield precious information about the application potential of the studied strain and for process design. It has indeed been shown that the kf value displays an optimum at an initial complex nitrogen concentration of 4.2% at a constant initial concentration of 7.5% lactose, while the value of kd was not significantly influenced by the amount of complex nitrogen (Degeest & De Vuyst, 1999). The application of a well-defined carbon/nitrogen ratio during the industrial process may hence lead to a boost of EPS levels. Eventually, mathematical information about the combined influence of several environmental factors on a biokinetic parameter of the model may then be

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combined by surface regression or by an approach based on the g-concept (Leroy et al., 2001). The g-function obtained through secondary modelling describes the response of the growth rate to changes in the environmental factor, with values ranging from 0 (no growth) to 1 (optimum growth). The g-concept states that certain environmental factors, such as pH, temperature, and water activity, act independently on the bacterial growth rate (Wijtzes, de Wit, Huis in ‘t Veld, van ‘t Riet, & Zwietering, 1995). Finally, a complex tertiary model may be conceived, taking into account the global effect of all concerned environmental factors and their interactions (Leroy and De Vuyst, unpublished results). After a validation procedure, this model may then be used to simulate bacterial functionality in a food environment. As an example, the HePS production in milk by the HePS-positive yoghurt starter cultures can be estimated by a simple modelling approach. In case of S. thermophilus LY03, a correlation between biomass formation and cumulative base consumption was obtained from fermentation experiments in MRS broth (Degeest & De Vuyst, 1999). Now, this correlation permits to estimate biomass (CDM) from base consumption during fermentations in milk, which may in turn be used to predict EPS yields using a mathematical model. Fig. 2 represents the simulation of biomass and HePS production by S. thermophilus LY03 based on the experimentally determined cumulative base consumption. At present, this model allows predicting the

metabolic behaviour of a HePS-producing starter culture during milk fermentations.

9. Process engineering of heteropolysaccharide production by lactic acid bacteria The HePS production by the LAB strains can hardly compete with aerobic bacteria, such as X. campestris, as is reflected in the production levels of xanthan gum (30– 50 g L1) compared to those of the HePS produced by the LAB (0.1–1.5 g L1). From an economic point of view, a tenfold increase in the HePS production by the LAB, to obtain 10–15 g L1, may be required to produce these HePS as a food additive. However, a much smaller amount should be enough to exploit in situ applications. 9.1. In vitro production of HePS in fed-batch cultivation Since the HePS production from the thermophilic LAB strains seems to be coupled to growth and since the LAB growth is presumably inhibited by the formation of lactate, there is scope for productivity improvement by reducing the concentration of lactate in the culture broth, either via fed-batch cultivation with cell recirculation or extractive fermentation. Although, neither higher biomass nor higher HePS concentrations were obtained through fed-batch fermentations using different feeding strategies (constant feeding rate and

Fig. 2. Modelling of cell growth (’, g CDM L1), substrate consumption (E, g lactose L1), excretion of galactose (n, g galactose L1), and the production of functional metabolites (m, g lactic acid L1; K, ½EPS in mg PDM L1) in enriched milk medium for the exopolysaccharide producer S. thermophilus LY03 at 421C and a controlled pH of 6.2. Lines are according to the model, symbols represent experimental data. The experimental values for cell growth are calculated from the cumulative base consumption using a second order correlation.

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acidification-controlled feeding rate) as compared to similar batch fermentations, a high lactose pressure during acidification-controlled fed-batch fermentations revealed a higher stability of the high-molecular-mass HePS from S. thermophilus LY03 upon prolonged fermentation (Vaningelgem, Degeest, & De Vuyst, unpublished results). Additionally, a controlled feeding strategy may perhaps make it possible to produce tailormade HePS without the need of manipulating the producer strain genetically.

milk, followed by an acidification step to allow clotting of the milk and to improve aroma and taste formation. The results demonstrated that the two-step fermentation process affords good bacterial growth, doubles HePS yields, and increases viscosity of the milk medium. Unfortunately, these increased HePS yields and viscosities are at the expense of the time needed to reach the maximum values. Interestingly, no syneresis was observed upon storage of the fermented milk as compared to the one-step fermentation process.

9.2. In situ production of HePS during yoghurt manufacture

Acknowledgements

Traditionally, yoghurt is produced by inoculation of milk with Lb. delbrueckii subsp. bulgaricus and S. thermophilus (in a 1 : 1 ratio) as starter cultures. Two basic types of yoghurt exist, according to its physical state in the retail container: set yoghurt and stirred yoghurt. Set yoghurt is fermented after being packed in a retail container, and stirred yoghurt is almost fully fermented in a fermentation tank before it is packed, the yoghurt gel being broken up during the stirring. To obtain the necessary thickening and texture, and to avoid water separation (syneresis), the milk is often fortified with milk solids through the addition of defatted milk powder, whey powder, and stabilizers (chemically modified starch, carrageenan, guar gum, pectin, gelatin, caseinate, etc.). However, fortification of the milk is not beneficial from an economic point of view. In addition, food additives may adversely affect the true taste and aroma of yoghurt. Moreover, their use results in a non-natural image, and is not allowed in all countries. The use of yoghurt starter cultures that contain strains that produce HePS is a promising alternative (Cerning, 1995; Bouzar et al., 1996; Cerning & Marshall, 1999; De Vuyst & Degeest, 1999; Marshall & Rawson, 1999; Rawson & Marshall, 1997; Ricciardi & Clementi, 2000). The HePS that are produced in situ, i.e. through inoculation of milk with a yoghurt starter culture that contains the HePS-producing strains, have indeed the capacity to retain water and hence avoid syneresis, to improve the viscosity and hence guarantee a good final texture, and to replace fat without affecting the mouth feel when the yoghurt is eaten (De Vuyst & Degeest, 1999; Marshall & Rawson, 1999). However, the HePS production in milk by the thermophilic LAB strains such as S. thermophilus is low and unstable when traditional batch process technologies for the production of yoghurt are carried out (De Vuyst & Degeest, 1999). Therefore, to exploit the HePS production capacity of a yoghurt starter strain maximally, a new, two-step, process technology for yoghurt production has been proposed (Degeest & De Vuyst, unpublished results), consisting of a first, HePS production step through on-line control of the pH of the inoculated

The authors’ research on the EPS production by the LAB was financially supported by the Institute Danone by means of a ‘Navorsingskrediet voor Fundamenteel Voedingsonderzoek’, the Institut Yoplait International, the European Commission (EU) (grants FAIR-CT984267 and IC15-CT98-0905), the Flemish Institute for Encouragement of Scientific and Technological Research in Industry (IWT), the Fund for Scientific Research (FWOFFlanders), the Link 2000 Action of the Brussels Capital Region, and the Research Council of the Vrije Universiteit Brussel (VUB). BDG and FV are recipients of an IWT fellowship.

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