Emerging fermentation technologies: Development of novel sourdoughs

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1 FOOD MICROBIOLOGY Food Microbiology 24 (2007) Emerging fermentation technologies: Development of novel sourdoughs G. Lacaze, M. Wick, S. Cappelle Puratos Group, BU Bioflavors, Industrialaan, 25, 1702 Groot-bijgaarden, Belgium Abstract The increasing knowledge of sourdough fermentation generates new opportunities for its use in the bakery field. New fermentation technologies emerged through in depth sourdough research. Dextrans are extracellular bacterial polysaccharides produced mainly by lactic acid bacteria (LAB). These bacteria convert sucrose thanks to an inducible enzyme called dextransucrase into dextran and fructose. The structure of dextran depends on the producing micro-organism and on culture conditions. Depending on its structure, dextran has specific properties which lead to several industrial applications in different domains. The use of dextran is not widely spread in the bakery field even if its impact on bread volume and texture was shown. A new process has been developed to obtain a sourdough rich in dextran using a specific LAB strain able to produce a sufficient amount of HMW dextran assuring a significant impact on bread volume. The sourdough obtained permits to improve freshness, crumb structure, mouthfeel and softness of all kinds of baked good from wheat rich dough products to rye sourdough breads. From fundamental research on dextran technology, a new fermentation process has been developed to produce an innovative functional ingredient for bakery industry. r 2006 Published by Elsevier Ltd. Keywords: Sourdough fermentation; Microbial exopolysaccharides; Dextran; Bread texture 1. Introduction Sourdough in bakery was traditionally used for its leavening effect (CO 2 production) and its aromatic impact on bread (organic acids and volatile compounds). Several studies on sourdough fermentation and its microbiology have shown that, besides organic acids and CO 2, other kinds of compounds are produced. Indeed, bacteriocins (Corsetti et al., 2004), antifungal compounds (Lavermicocca et al., 2003) or exo-polysaccharides (Tieking et al., 2003) are released during the fermentation. Furthermore, several degradation processes can occur. For example, Gliadin-like fractions of gluten (Rollan et al., 2005) are damaged thanks to the microbiological activity. These researches open new perspectives for the use of sourdough fermentation in baked products, extending its applications to domains such as health and pleasure. These new findings lead also to emerging fermentation technologies. Dextran technology is a good example of such an emerging fermentation technology. Indeed, from the Corresponding author. address: scappelle@puratos.com (S. Cappelle). isolation of one specific strain producing dextran to the production of sourdoughs based on dextran, a great work of fermentation development and optimization has been done. 2. Dextran structure, microbial synthesis and dextran properties The term dextran is a collective term given to a group of bacterial polysaccharides. The basic structure of dextran molecules are (1 6) linked a-d-glucopyranosyl residues. Sometimes, the linear backbone contains branchings at C- 2, C-3, or C-4. Isolated (1 3) linked a-d-glucopyranosyl residues or sequences of these residues can interrupt the (1 6) regions. All of the dextrans are more or less ramified (Fig. 1), and the branching very much depends on the subspecies (Jeans et al., 1954). Most of the branchings are composed of single a-d-glucopyranosyl residues, although branchings have been found with 2 50 monomers. Some branchings are formed by (1 3) linked a-d-glucopyranosyl residues. The structure such as length of the chain, degree of branching, type of links of the dextrans mainly depends on the producing micro-organism. For example, /$ - see front matter r 2006 Published by Elsevier Ltd. doi: /j.fm

2 156 ARTICLE IN PRESS G. Lacaze et al. / Food Microbiology 24 (2007) Fig. 1. Structure of fragment of dextran molecule. nc 12 H 22 O 11 (C 6 H 10 O 5 )n + nc 6 H 12 O 6 sucrose dextran fructose Fig. 2. The conversion of sucrose to dextran by dextransucrase. Leuconostoc mesenteroides NRRL B512(F) produces dextran containing 95% of a-1,6 bonds and 5% of a-1,3 bonds. Dextrans are synthesized from sucrose through the production of an inducible dextransucrase (1,6-a-D-glucan 6-a-D-glucosyltransferase, EC ). This enzyme, usually extracellular, is produced from a variety of different micro-organisms. These bacteria mainly belong to the group of lactic acid bacteria, and more specifically to the species of the Lactobacillus, Leuconostoc and Streptococcus genus. Certain bacterial strains have been shown to produce dextrans of various structures, and this was attributed to the excretion by the micro-organism of different dextransucrases (Coˆte and Robyt, 1982). Dextrans can also be synthesized by means of dextrine dextranase of, for instance, Acetobacter capsulatus ATCC (Yamamoto et al., 1993). Most microbial polysaccharides are the products of intracellular catabolic conversion of substrate to intermediates and finally to polymer. For dextrans, this is not the case. Indeed, the substrate, sucrose, does not enter the micro-organism cell. In excess of sucrose, the cell release dextransucrase which converts sucrose to dextran and fructose as shown in Fig. 2. During the enzymic reaction, dextran is linked to the enzyme at the reduction end and glucose units are added at the reducing end by insertion between the dextransucrase and the growing dextran chain (Robyt et al., 1974).When sucrose is depleted, the bacteria consume the fructose which has been accumulated during the polymerization process (Neely and Nott, 1962). Dextransucrase catalyses the transfer of the glucosyl unit of sucrose to different acceptor molecules which are normally the growing dextran chain. When another substrate acceptor is also present, oligosaccharides are produced and part of the glucosyl moieties from glucose is consumed to form these acceptor products (Robyt and Walseth, 1978), decreasing the dextran yield. Thus, as the ratio of acceptor to sucrose increases, the proportion of dextran decreases and that of oligosaccharides increases. Over thirty different carbohydrates are known to act as acceptors of varying activities (Robyt and Eklund, 1982). The most efficient acceptor molecule is maltose followed in order by isomaltose, nigerose, a-methyl-d-glucopyranoside and D- glucose. In presence of maltose, dextransucrase synthesized panose (6,2-a-D-glucopyranosyl maltose) and a homologous series of 6,2-isomaltodextrinosyl maltoses (Fu and Robyt, 1990). Dextrans molecular weights range from to and even higher. The molecular weight of the dextran can be affected by the presence of acceptors (as explained above). Temperature, sucrose concentration and enzyme concentration have also been reported as affecting the molecular weight of dextran (Tsuchiya et al., 1952). Thus, at low sucrose concentrations (10%), dextrans of high molecular weight are formed (410 8 daltons). Dextrans solubility depends on their branched structure. Soluble dextrans are composed of sequences of (1 6) linked a-d-glucopyranosyl residues, on which, at irregular intervals, branchings of single a-d-glucopyranosyl residues are substituted. Insoluble dextrans are more complex and they often contain more (1 3) linked a-d-glucopyranosyl sequences. Dextrans form viscous solutions. These solutions show a Newtonian behaviour when concentrations o30% w/w for low molecular weight dextrans. Dextrans with a higher molecular weight show a slightly pseudoplastic behaviour when concentrations 41.5% w/w. There are no single correlations between the viscosity of dextrans solution and its branchings. The viscosity enhancing effect is due to a combination of structure (more or less branched) and molecular weight (Jeans et al., 1954). 3. Industrial use of dextran The main applications for dextran are in the pharmaceutical and medical sectors. The low molecular weight dextran is used as a blood plasma substitute as solutions containing 6% of these dextrans have viscosity value similar to human blood plasma. Moreover, derivatives of dextran, like dextran sulfate, are used as a substitute for heparin. Sephadex, which is a commercial gel filtration material, is made of epichlorohydrin cross-linked dextran. Although dextran can be used in food products as a conditioner, stabilizer, or bodying agent, it is rare to find it as a commercial product in the food industry. The use of dextrans in the field of bakery is not widely spread, although a number of applications have been described. For example, the US Patent (Bohn, 1961) describes the incorporation of a sufficient amount of dextrans in bakery products to soften the gluten content and to increase the specific volume. This document describes that the bread which contains dextran was about 20% bigger in volume than products which do not contain dextrans. In this particular example, said dextrans are prepared by growing the micro-organism Leuconostoc mesenteroides B512, resulting in dextrans having a molecular weight from about to about daltons.

3 G. Lacaze et al. / Food Microbiology 24 (2007) According to these facts, it has been decided to develop sourdoughs based on dextran in order to propose to bakers a solution to improve texture/volume of their baking products. 4. Industrial technology for producing dextran based sourdoughs The first step consisted in selecting a strain able to produce sufficient amounts of dextran to get a positive impact on bread quality. Strains were first isolated from sourdoughs and cultured milk products and then selected on solid modified MRS medium supplemented with sucrose (4%). Ropy colonies were retained for dextran production which was done in two steps approach: enzyme production then bioconversion. This 2 steps process consisted in applying different sucrose concentrations and ph. A strain, isolated from a natural sourdough system (Panettone) and identified as a L. mesenteroides spp. was selected. This strain (deposit number LMGP-16878) produces a dextran which gave significant volume improvement in wheat bread baking trials compared to guar and dextran from L. mesenteroides B512F (Table 1). These results clearly show that the type and the origin of the polymer is critical. The dextran synthesized by the strain LMGP is characterized by its structure 95.4% of a-1,6 bonds and 4.6% of a-1,3 bonds and its high molecular weight. These data show that dextran with a high molecular weight and a linear chain structure is more efficient on bread volume than dextran with a high molecular weight and more branching. The second step was to develop a specific sourdough fermentation using the strain selected first, in order to obtain effective amount of dextran in the sourdough. These sourdoughs, one based on wheat flour and the other based on rye flour, are obtained by the fermentation of flour and sucrose by L. mesenteroides LMGP The fermentation, carried out at 25 1C, is produced in two stages. The first stage consists in the production of the starter culture L. mesenteroides LMGP on an adapted growth medium. In a second stage, dough composed of flour, water and sucrose is started with the strain LMGP and fermented at 25 1C. The optimum sucrose concentration and fermentation process Table 1 Effect of addition of dextrans produced by different strains on final bread volume and comparison with guar gum (European patent EP B1) Reference+0.5% guar 10 Reference+1% guar 5 Reference+0.5% dextrans B512F 8 Reference+1% dextrans B512F 12 Reference+0.5% dextrans LMGP Reference+1% dextrans LMGP Volume increase compared to reference (vol%) parameters were determined. During fermentation, the ph drops naturally through the organic acid production of the strain, passing all ph levels of enzyme induction, bioconversion and fermentation of residual fructose (Lazic et al., 1993; Tsuchiya et al., 1952). The increase of dough viscosity during this step demonstrates the production of dextran. The final wheat sourdough is characterized by a ph comprised between 3.5 and 3.9, an acidity around 40 ml of NaOH 0,1N /10 g, and a viscosity between 4000 and 5500 cp. The rye sourdough has a ph between 3.2 and 3.6 and an acidity around 49 ml of NaOH 0.1N /10 g. These sourdoughs are used at a level of 10% on flour weight and improve volume and texture of the baked products. 5. Applications of dextran in bakery The advantages of this specific type of dextran and the dextran containing sourdoughs in bakery applications are multiple. More water can be bound in the dough: as dextran is a hydrocolloid, it can bind high amounts of water. Higher dough yield often leads to an improved freshness of the end product. Dextran improves dough stability and gas retention through a structure build up of the dextrans and interaction with gluten network. In Fig. 3, it is clearly shown that dextrans build up a certain structure when a shear force is applied on a 1% solution. This is not the case with the well-known gums like guar and xanthan. This is probably explained by the fact that the long linear dextran molecules line up very well and hydrogen interactions occur. The oven jump is significantly better after addition of dextran. This can also be explained due to the improved dough stability and gas retention. The crumb structure is altered. The gas bubbles are more long shaped, typical for long fermentation processes. Mouthfeel is improved: a moister mouthfeel is obtained by addition of dextran. The effect becomes even clearer the longer the bread is conserved. In a lot of applications, a very short bite can also be experienced after addition of a dextran containing sourdough. The softness of the bread crumb is measured viscosity (Pa.s) guar xanthan dextran time (minutes) Fig. 3. Improved dough stability due to structure build up of dextran.

4 158 G. Lacaze et al. / Food Microbiology 24 (2007) with a texture analyzer. All of these advantages are described in the patent EP B1 (Vandamme et al., 1997). The application fields of dextran containing sourdoughs are multiple: from rich wheat doughs (fat, sugar) up to very heavy rye dough systems (rye breads). In viennoiserie, the effect of addition of sourdough has been evaluated in industrial trials (milk bread application). Reference milk bread (without sourdough) has been compared with a milk bread containing 5% of a liquid active wheat sourdough based on dextran. After 2 weeks conservation, hardness, ph and water activity have been measured. It has been shown that hardness was significantly reduced with liquid wheat dextran sourdough (Fig. 4). ph of milk bread crumb was reduced by 0.3 units with the addition of sourdough. Water activity was identical. A panel test run on 40 people showed that the milk bread that contained dextran sourdough was significantly preferred to the reference. Moreover, a consumer panel test has been run on 205 persons in Nantes (France). Those people were chosen at the exit of a supermarket but no selection was done on people (age, profession, etc.). Butter brioches containing wheat dextran sourdough were compared with brioches without, in a preference tests. People were asked to rank the product in terms of general preference, by giving a score from 1 to 10. The sum of rank is calculated at the number of people multiplied by the Hardness (g) REFERENCE WITHOUT SOURDOUGH 5% LIQUID WHEAT SOURDOUGH BASED ON DEXTRAN Essais Fig. 4. Effect of liquid dextran wheat sourdough on hardness of milk bread after 2 weeks conservation Sum of ranking Butter brioche Butter brioche with 5% dextran sourdough Sum of ranking Fig. 5. Effect of dextran sourdough addition in butter brioches on consumers preferences.

5 G. Lacaze et al. / Food Microbiology 24 (2007) score they gave. It appeared that the brioches containing dextran sourdough were significantly preferred to the reference as shown in Fig. 5. In rye mixed bread, the influence of the addition of dextran rye sourdough on the aging of rye mixed bread was evaluated. Softness measurements were done with a texture analyzer. The softer the bread, the less force the machine needs to put on the crumb to cause a determined deformation of the crumb. In parallel, evaluation of freshness was done by tasting the bread. The results in the mixed rye bread are shown in Fig. 6 and are expressed as decrease in hardness of the bread containing dextran. Softness and moistness of the crumb of baked products has been improved both after 4 and 7 days. More remarkably, the difference between 4 and 7 days is lower when using dextran sourdough, which shows that also staling of the crumb is delayed. In these breads, volume and specific volume (volume divided by weight) were also measured. Bread volume is measured in a typical bakery apparatus filled with sesame seeds. Bread volume is deduced from a Hardness (g) Hardness after 4 days Hardness after 7 days Liquid sorudough Liquid sourdough based on dextran Fig. 6. Hardness measurement in rye breads containing liquid sourdough or liquid sourdough based on dextran % + 25 % Specific volume Liquid rye sourdough Liquid rye sourdough based on dextran Liquid rye sourdough based on dextran + Rye bread mixe Series Fig. 7. Effect on dextran sourdough and bread mix on specific volume increase in rye breads.

6 160 ARTICLE IN PRESS G. Lacaze et al. / Food Microbiology 24 (2007) standard volumes occupied by sesame seeds. It has been shown that addition of liquid rye sourdough based on dextran has a significant effect on volume increase and specific volume increase. This effect was even higher when combined with an enzymic bread mix (Fig. 7). 6. Concluding remarks From the isolation of specific bacterial strain in Panettone, it has been possible to develop an innovative functional bakery ingredient. Strain selection for dextran sucrase production and sourdough fermentation have been done at an industrial level. Significant effects for use of dextran sourdough in bakery application were shown. Bread volume is increased and crumb softness is improved. This has been shown in rye breads application, viennoiserie (rich doughs), as well as in wheat breads application (data not shown). Dextran sourdoughs, based on wheat or rye, are typical examples of emerging fermentation technology and can be used in various bakery applications. Improvement of flavor, softness and freshness answers one of the main concerns of the bakery industry which is to create baked products with outstanding organoleptic quality for all consumers. References Bohn, R.T., Addition of dextran to bread doughs, US Patent Corsetti, A., Settanni, L., Van Sinderen, D., Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity. J. Appl. Microbiol. 96, Coˆté, G.L., Robyt, J.F., Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B that synthesizes alternating (1 6), (1 3)-a-D-glucan. Carbohydr. Res. 101, Fu, D., Robyt, J.F., Acceptors reactions of maltodextrins with Leuconostoc mesenteroides B-512FM dextransucrase. Arch. Biochem. Biophys. 283, Jeans, A., Haynes, W.C., Williams, C.A., Rankin, J.C., Melvin, E.H., Austin, M.J., Cluskey, J.E., Fisher, B.E., Tsuchiya, H.M., Rist, C.E., Characterization and classification of dextrans from ninety-six strains of bacteria. J. Am. Chem. Soc. 76, Lavermicocca, P., Valerio, F., Visconti, A., Antifungal activity of phenyllactic acid against molds isolated from bakery products. Appl. Environ. Microbiol. 69, Lazic, M.L., Veljkovic, V.B., Vucetic, J.I., Vrvic, M.M., Effect of ph and aeration on dextran production by Leuconostoc mesenteroides. Enzyme Microb. Technol. 15, Neely, W.B., Nott, J., Dextransucrase, an induced enzyme from L. mesenteroides. Biochemistry 1, Robyt, J.F., Eklund, S.H., Stereochemistry involved in the mechanism of action of dextransucrase in the synthesis of dextran and the formation of acceptors products. Bioorg. Chem. 11, Robyt, J.F., Walseth, T.F., The mechanism of acceptor reactions of Leuconostoc mesenteroides B512(F) dextransucrase. Carbohydr. Res. 61, Robyt, J.F., Kimble, B.K., Walseth, T.F., The mechanism of dextransucrase action: direction of dextran biosynthesis. Arch. Biochem. Biophys. 165, Rollan, G., De Angelis, M., Gobbetti, M., de Valdez, G.F., Proteolytic activity and reduction of gliadin-like fractions by sourdough lactobacilli. J. Appl. Microbiol. 99, Tieking, M., Korakli, M., Ehrmann, M.A., Gänzle, M.G., Vogel, R.F., In situ production of exopolysaccharides during sourdough fermentation by cereal and intestinal isolates of lactic acid bacteria. Appl. Environ. Microbiol. 69, Tsuchiya, H.M., Koepsell, H.J., Corman, J., Bryant, G., Bogard, M.O., Feger, V.H., Jackson, R.W., The effect of certain cultural factors on the production of dextransucrase by Leuconostoc mesenteroides. J. Bacteriol. 64, Vandamme, E.J., Renard, C.E.F.G., Arnaut, F.R.J., Vekemans, N.M.F., Tossut, P.P.A., Process for obtaining improved structure buildup of baked products, EP B1. Yamamoto, K., Yoshikawa, K., Okada, S., Effective dextran production from starch by dextrin dextranase with debranching enzyme. J. Ferm. Bioeng. 76,

ON THE DIFFERENCE IN ADSORPTION ON SEPHADEX GEL OF THE DEXTRANSUCRASE OF STREPTOCOCCUS BOVIS GROWN ON SUCROSE AND GLUCOSE MEDIA

J. Gen. App!. Microbiol., 34, 213-219 (1988) ON THE DIFFERENCE IN ADSORPTION ON SEPHADEX GEL OF THE DEXTRANSUCRASE OF STREPTOCOCCUS BOVIS GROWN ON SUCROSE AND GLUCOSE MEDIA TOSHIRO HAYASHI, RYO IOROI,*