Screening methods for the proteolytic breakdown of gluten by lactic ...

Eur Food Res Technol (1999) 209 : 428–433

Q Springer-Verlag 1999

ORIGINAL PAPER

Karina Wehrle 7 Noeleen Crowe 7 Ineke van Boeijen Elke K. Arendt

Screening methods for the proteolytic breakdown of gluten by lactic acid bacteria and enzyme preparations

Received: 25 February 1999

Abstract Gluten is the major protein in wheat and it is largely responsible for the rheological characteristics of bread dough. The sourdough process is a traditional dough fermentation process with lactic acid bacteria. The proteolytic activity of lactic acid bacteria derived from starter cultures used in sourdough or meat fermentation was tested on gluten as a substrate. The activities of commercial enzyme preparations, which were derived from bacterial or fungal sources, were also evaluated. Proteolytic breakdown of gluten protein in an agar medium by either bacterial culture or enzymes was evident by a clear zone around wells after staining with Coomassie blue. The increase in TCA-soluble material due to gluten breakdown was measured spectrophotometrically. A third test evaluated the release of free amino acids due to exoproteolytic activity. The agar based test showed positive results for the commercial enzyme preparations and one Micrococcus strain. All sourdough strains were positive in the enzyme test whereas some starters from the meat fermentation process were not able to break down gluten under test conditions. Variations in the release of free amino acid indicated differences in the enzyme systems of the lactic acid bacteria tested. Key words Proteolysis 7 Gluten 7 Sourdough 7 Starter 7 Lactic acid bacteria

K. Wehrle Department of Food Science and Technology and National Food Biotechnology Centre, University College Cork, National University of Ireland, Cork, Ireland N. Crowe 7 I. van Boeijen 7 E.K. Arendt (Y) Department of Food Science and Technology, University College Cork, National University of Ireland, Cork, Ireland e-mail: e.arendt6ucc.ie

Introduction Enzymes play a major role in the production of bakery products. The innate enzyme activity of the raw material is very important in the quality classification of wheat. Excessive enzyme activity, which is found in sprout-damaged wheat, could lead to the rejection of a whole batch. On the other hand, controlled addition of exogenous enzymes provides an excellent method of improving the processing performance of flour and the quality of the final products [1]. From the technological viewpoint carbohydrate-degrading enzymes were regarded for a long time as the technologically most important enzymes in bread production [2]. Fermentable sugars, which are the products from amylase activity, are essential for the leavening process by yeast fermentation. The application of proteases gained more importance with the introduction of high-speed mechanised bread production lines. The modification of the gluten network improves the dough’s machinability. Additionally it affects aroma, crust colour and crumb texture of the final products [3]. It has been shown that even small amounts of proteolytic enzymes greatly affect the physical properties of gluten [4]. Sources of exogenous enzymes are mainly selected bacteria or fungi strains. It is also known that lactic acid bacteria, which are used in the sourdough process, show proteolytic activity [5]. To date the selection criteria for sourdough bacteria have been focused mainly on the development of a preferable aroma; enzyme activity and the effects on the rheological properties of the dough have not been monitored to date. The most common method for measuring proteolytic activity is spectrophotometrically; it is based on the increase in TCA-soluble material during exoproteolytic breakdown [6]. Casein or haemoglobin are widely used as substrates for these tests [7], their advantages being that they are non-toxic, widely available and at least partially soluble in a buffer. However, results obtained from these tests do not reflect the conditions that are

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present in a dough system with gluten as the main protein source. The activity and pH optimum for proteolytic activity in flour vary widely depending on the substrate used [8]. Proteolytic activity in sourdoughs has been detected by measuring the increase in free amino acids during fermentation of sourdough in comparison with dough systems without starter cultures [9–11]. The concentration of some amino acids increased during the sourdough process, whereas the concentration of others decreased. The metabolically active lactobacilli and yeasts in the dough multiplied and consumed some of the amino acids. No distinction could be made between the breakdown of soluble proteins and the modification of gluten protein when this method was used. The aim of this project was the development of rapid methods for the evaluation of proteolytic activity of sourdough bacteria on gluten. The method developed is also applicable to the testing of purified enzymes and commercial enzyme preparations.

Materials and methods Strains. The strains tested were either obtained from wheat or rye sourdough starters used in commercial bread production or derived from starter cultures used in meat fermentation (Table 1). Enzymes. Two commercially available proteases were used, Bioprotease P concentrate and Bioprotease NICOL (Quest, Ireland). Bioprotease P concentrate is offered to the baking industry to achieve better dough extensibility; it is derived from a selected strain of Aspergillus oryzae and has an activity of 400,000 haemoglobin units/g. This enzyme was used in the plate tests at concentrations of 1% and 10% in water. Bioprotease N100L is derived from a selected strain of Bacillus subtilis and is mainly used in the brewing industry; its activity level is a minimum of 100,000 Nitrogen Producing Units (NPU)/ml. This liquid enzyme preparaTable 1 Bacterial cultures Strain

Short name

Application

Source 2a

Lactobacillus delbrukii L. sanfrancisco (1) L. sanfrancisco (2) L. sanfrancisco (3) L. plantarum L. brevis L. pentosus LP1 L. pentosus 03 A L. pentosus olivarum L. sake LS25 L. curvatus L. alimentarius BJ33 Pediococcus pentosaceus PC1 P. acidilactici Lactococcus lactis Micrococcus varians

L. delb L. sanf 1 L. sanf 2 L. sanf 3 L. plant L. brev L. pent 1 L. pent 03A L. pent oliv L. sake L. curv L alim P. pent

Sourdough Sourdough Sourdough Sourdough Sourdough Sourdough Meat Meat Meat Meat Meat Meat Meat

Bö Bö Bö Bö Bö Bö CH RM RM G G CH CH

P. acid Lc. Lactis M. var

Meat Meat Meat

CH Ne

a Bö Böcker Sauerteigprodukte, Minden, Germany; CH Christian Hansen, Hørsholm, Denmark; RM Rudolf Müller, Pohlheim, Germany; G Gewürzmüller, Stuttgart, Germany; Ne Nestec, Lausanne, Switzerland

tion was used in pure form and in a 1% water solution for the plate tests. In the spectrophotometric tests, both enzymes were used in 0.05% concentrations. Chemicals used for the growth media, plate media and solutions were obtained from the following companies: MRS broth, meat extract (Oxoid, Basingstoke, UK); tryptone, yeast extract, agar (Difco, Detroit, Mich., USA); NaCl, glucose, trisodium citrate, citric acid monohydrate, Na2HPO472H2O, Coomassie brilliant blue R-250, acetic acid, trichloracetic acid (British Drug House, Poole, UK); maltose, CaCl272H2O, methanol (Merck, Darmstadt, Germany); fructose, sodium azide, Tris-HCl, sodium acetate, cadmium chloride ninhydrin, (Sigma, St. Louis, Miss., USA); ethanol 99.5% (Carbery Distilleries, Ireland). Gluten preparation. Gluten was separated from wheat flour using a Glutomatic 2000 System (Falling Number, Stockholm, Sweden) according to the International Association for Cereal Science and Technology standard method for determination of wet gluten [12]. The gluten proteins were separated from wheat dough by a standardised washing procedure with a 2% NaCl solution. After centrifugation the wet gluten was freeze-dried and ground in a mill. Ground gluten was stored at 4 7C in a sealed container until use. Microorganism growth conditions. Lactic acid bacteria derived from sourdough were cultured in a modified MRS medium [13] to meet their particular requirements for growth factors. Lactic acid bacteria from the meat fermentation process were grown in MRS broth. PM broth was used to grow Micrococcus varians. All strains were grown at 30 7C for 24 h. Plate test. Media for the plate test contained either gluten or casein as protein source. A basic medium with 1.5% agar, 1% protein, 0.56% tryptone, 0.25% yeast extract, 0.1% glucose, 0.44 g trisodium citrate and 0.2% CaCl2 was prepared [14]. Variations of the basic medium involved the replacement of trisodium citrate with a citric acid/Na2HPO4 buffer in two different concentrations to achieve pH values of 4 (13 g/l citric acid monohydrate and 13.5 g/l Na2HPO472 H2O) and 7 (4.0 g/l citric acid monohydrate and 28.8 g/l Na2HPO472 H2O). To avoid hydrolysis of the agar during autoclaving at a pH of 4, the buffer solution and agar were autoclaved separately and mixed aseptically when cooled down. While preparing the medium containing gluten and during pouring of the plates, the medium needed to be constantly stirred to avoid the formation of a single gluten ball. Due to the insolubility of gluten in the media smaller agglomerates of protein remained visible in the plates. Constant agitation led to a homogeneous dispersion of the particles. After solidification, three wells (6 mm diameter) were punched into each plate. The wells were inoculated with 20 ml cell suspension or enzyme solution and incubated for 48 h at 30 7C. After 48 h the agar layer was stained for 5 min in a solution of 5 g Coomassie brilliant blue R-250, 500 ml methanol and 92 ml acetic acid in 1 l distilled water. Subsequently the plates were destained overnight in a solution of 250 ml ethanol and 50 ml acetic acid in 1 l of distilled water. The plates were evaluated by measuring the diameter of clear zones formed around the wells. Spectrophotometric method. Proteolytic breakdown of protein was measured as the increase in absorption with time in a TCA solution. Twenty-five millilitres of solution I [(0.5% (v/v) 1 M Tris-HCl, 0.2% (v/v) 4% sodium azide, 0.074% (w/v) CaCl272 H2O, 0.8% protein (either casein or gluten)] was inoculated with 1 ml of either cells or enzyme. The cells were harvested after 24 h growth, in a stationary stage, by spinning down 15 ml of culture at 3,000 rpm for 15 min (Centrifuge Hermle 2360, Berthold Hermle, Gosheim, Germany). The pellet was re-suspended in 1 M Tris-HCl buffer, the total cell count for each culture was about 1.5!10 210. The incubation period for the enzyme test took up to 6 h in a shaking waterbath at 30 7C. The enzymatic reaction was stopped by adding 1 ml of a solution containing 1.634% (w/v) trichloracetic acid 1.804% (w/v) sodium acetate and 1.886% (v/v)

430 Table 2 Zones of proteolytic activity on agar plates (LAB lacticacid bacteria, – not tested)

Sample

Casein pH 7

Gluten pH 7 basic

Gluten pH 7

Gluten pH 4

Sourdough LAB Meat LAB Micrococcus varians Enzyme bacterial (1%) Enzyme bacterial (pure) Enzyme fungal (1%) Enzyme fungal (10%)

0a 0 19 30 45 33 39

0 0 19 22 32 15 31

0 0 – 15 30 18 17

0 0 – 0 0 15 24

2a

All values are the average diameters of three zones in millimetres

acetic acid. The samples were left at room temperature for 30 min. Subsequently the samples were centrifuged at 14,000 rpm (Eppendorf centrifuge 5415C, Engelsdorf, Germany). The absorption of the supernatant was measured at 275 nm in a quartz cuvette, with a light path of 1 cm in a Beckman DU 640 spectrophotometer (Beckman, Fullerton, Calif., USA). Enzymatic breakdown was measured over periods of 6 or 3 h, samples being taken every hour. Each sample was measured in triplicate. The readings for absorption of a blank containing 1 ml of 1 M Tris-HCl buffer instead of the culture or enzyme solution was subtracted each time from the values for the samples. Absorptions at time zero were subtracted from the following readings for each measurement to standardise all graphs. Incubating the cell suspension in solution I containing no protein tested autolysis of the cells. Any increase in absorption of this test series would indicate the release of TCA-soluble material from the cells. Determination of free amino acids. The release of free amino acids due to exoproteolytic activity was measured with a colorimetric method using a cadmium-ninhydrin reagent. Cadmiumninhydrin reacts with the amino group of free amino acids and forms a complex with an absorption maximum at 507 nm. The cells or enzymes were incubated for up to 6 h as described for the spectrophotometric method. At time zero, 10 ml of the sample was centrifuged for 15 min at 3000 rpm (Hermle 2360). The supernatant (1 ml) was added to 2 ml of the cadmium-ninhydrin reagent (0.8 g cadmium-ninhydrin dissolved in 80 ml ethanol and 10 ml acetic acid, with 1 g CaCl2 diluted in 1 ml distilled water added) and heated at 84 7C for 5 min. After cooling down, the absorption was measured at 507 nm in a 1-cm cuvette with a spectrophotometer (Beckman DU 640), and the absorption of a blank containing ninhydrin reagent and 1 ml distilled water was subtracted. The difference in absorption between time 0 and 6 h was calculated. The release of free amino acids from the cell material in the absence of gluten protein was measured by incubating cells

Fig. 1A,B Proteolytic activity of bacterial-derived baking enzyme on agar containing spray dried casein. A 1% solution B Pure solution

with solution I containing no protein. The release of free amino acids from gluten without the proteolytic activity of either microorganisms or enzymes was measured by incubating the gluten protein solution with Tris-HCl buffer and without cells.

Results Plate assay The incorporation of gluten into the agar medium resulted in plates with homogeneously distributed gluten particles of diameters less than 1 mm. Plates containing casein as protein source appeared opaque without any visible particles. After staining and subsequently destaining, both types of plate appeared completely blue with dark spots on the gluten plate. Both commercial enzymes showed positive results on the plates containing gluten and casein respectively at pH 7 (Table 2). The diameters of the zones were larger for the higher enzyme concentrations (Fig. 1). The diffusion distance of an enzyme depended on its concentration in the wells. Whereas the clear zones of the casein plates appeared completely transparent, some blue spots remained in the gluten plates, indicating large gluten particles were not degraded (Fig. 2). The formation of larger gluten agglomerates prevented close contact between enzyme and substrate and consequently no breakdown occurred. The diameters of the zones were smaller on the gluten plates than on the ca-

431 Fig. 2 Proteolytic activity on agar with gluten on A 10% solution of fungal derived enzyme and B Micrococcus varians

sein plates. The bacteria-derived enzyme did not show any breakdown at pH 4. It has been reported that bacteria-derived proteolytic enzymes are stable at a pH range of 5.5–10.5, whereas fungus-derived enzymes are stable at pH values down to 4.5 [8]. In the agar plate environment the fungal enzymes showed activity even at a pH of 4. Lactic acid bacteria derived from either meat or sourdough fermentation did not show any clear zone on any of the plates. Micrococcus varians created clear zones on both proteins (Fig 2). Spectrophotometric enzyme test Values of absorption for solutions containing gluten were initially higher than those for solutions containing casein as protein source. Isolated gluten protein already contained some TCA-soluble protein. During incubation the solution containing gluten but no enzyme or strain increased in average by 0.05 units over the 6-h period (Fig. 3). This increase was due to autolytic breakdown of gluten by attached enzymes from the flour, activity from microbial contamination or chemical hydrolysis. It has been reported that in dough many proteolytic enzymes are attached to the gluten but also that they are not very stable and can therefore easily be

Fig. 3 Increase in TCA-soluble material due to breakdown of substrate or strain. x 0.8% Gluten, l 1.6% gluten, g 0.8% casein, x 1.6% casein, without protein, X L. delb, } L. sake. See Table 1 for the full names of the bacteria

destroyed during gluten isolation [15]. However, for testing bacterial strains or exogenous enzymes the minor activity was preferable. Absorption did not increase in solutions containing casein as protein substrate. Freeze-dried casein did not show any autoproteolytic activity. Subtracting the blank values from the readings for the samples levelled the effect of the substrate. Incubation of L. sake (Table 1) without a protein substrate resulted in no increase of absorption, whereas incubation of the strain L. delb. (Table 1) caused an increase of 0.1 units within the 1st h of incubation. This increase was due to proteolytic breakdown of remaining media or the release of soluble material from the cells. Considering these results, proteolytic activity of the strains on gluten protein was evident with an increase in absorption of more than 0.1 units. The strains L. sake, L. curv., L. alim., Lc. lactis and M. var. (Table 1) did not show proteolytic breakdown on gluten as protein source (Fig. 4). All L. pent. strains (Table 1) and the two Pediococci strains were proteolytically active on gluten; the highest activities were found for L. pent. 03A (Table 1). All strains derived from sourdough starter cultures showed an increase in absorption when incubated with gluten (Fig. 5) as well as with casein (Fig. 6). The increase in absorption con-

Fig. 4 Proteolytic activity of lactic acid bacteris derived from meat starter cultures and M. varians on gluten. g L. sake, l L. curv, x L. alim, x Lc. lactis, c M. var, X L. pent 1, * L. pent 03A, [ L. pent oliv, } P. pent, L P. acid. See Table 1 for the full names of the bacteria

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Fig. 5 Proteolytic activity of lactic acid bacteria derived from sourdough starter cultures on gluten, g L. delb., l L. sanf. 1, L. sanf. 2, x L. sanf. 3, c L. plant., L. brev. See Table 1 for the full names of the bacteria

tinued for some strains over the whole incubation period (L. brev.) whereas for others absorption remained stable after the first 2 h of fermentation. In enzyme kinetics there are several reasons for this behaviour. Breakdown or inactivation of the enzyme itself led to the continuous reduction in enzyme activity with time. This effect was more pronounced with highly purified enzymes which could become unstable. Decreases in activity were also detected when the substrate was used up or the enzyme was inhibited by a high product concentration. A test with double substrate concentration did not further increase the absorption (data not shown), so the increase in TCA-soluble material was not limited by the substrate concentration rather than the enzyme concentration and activity. The commercial enzyme preparations showed a linear increase in absorption over the whole incubation time of 6 h with gluten, with the slope of the increase being larger for the fungus-derived enzyme than for the bacterial enzyme (Fig. 7). Much higher values for absorption were found for the test performed with the enzymes on casein substrate (Fig. 8). During the first 2 h of incubation the absorption increased sharply and remained stable thereafter. The enzyme test on gluten

Fig. 7 Proteolytic activity of enzyme preparations on gluten. [ Fungal, l bacterial, papain

Fig. 8 Proteolytic activity of enzyme preparations on casein. ([ Fungal, l bacterial, papain, filled symbols 1.6% casein, empty symbols 0.8% casein)

showed clearly that the activity of the enzymes remained stable over the whole test period. Incubation of the enzymes with twice the amount of casein resulted in a doubling of the values of absorption within a 2 h incubation time. This behaviour indicated that, in the case of the highly concentrated commercial enzymes, the enzymatic reaction was determined by the concentration of substrate. After 2 h of fermentation the concentration of product in relation to substrate suppressed the enzymatic reaction and absorption remained constant. Free amino acids

Fig. 6 Proteolytic activity of lactic acid bacteria derived from sourdough starter cultures on casein. g L. delb., l L. sanf. 1, L. sanf. 2, x L. sanf. 3, c L. plant., L. brev. See Table 1 for the full names of the bacteria

The treatment of gluten in the test solution over a period of 6 h without a strain or an enzyme did not result in any increase in free amino acids. The incubation of gluten with the sourdough strains led to the release of 1–8 mg lysine/g gluten (Table 3). The strains M. var. and L. sake did not show any exoproteolytic activity. In the previous test measuring the increase in TCA-soluble material these strains had also proved to be proteolytically in active. The enzyme derived from Bacillus subtilis produced an increase in free amino acids. An extremely large increase in free amino acids was observed for the fungal enzyme with an increase in absorption equivalent to the release of 30 mg lysine/g

433 Table 3 Release of free amino acids from gluten due to exoproteolytic activity Sample

mg lysine/g gluten

L. delb L. sanf 1 L. sanf 2 L. sanf 3 L. plant L. brev L. sake M. var Enzyme fungal Enzyme bacterial

5 7 8 2 7 1 –1 –1 8 30

gluten. The fungus-derived enzyme contained a considerable amount of exoproteolytic enzymes.

Discussion The modified plate test with gluten as protein source proved to be a suitable method for the evaluation of concentrated enzyme preparation and bacterial strains with a high extracellular proteolytic activity. Variations in the buffer system used in the plates allowed the testing of activity at different pH values to meet the conditions in specific dough systems. The test was not suitable when the enzymes were either intracellular or in any way connected to the cells so that they were not able to diffuse into the media and form clear zones. Negative results from this test, which were found for all the sourdough strains, did not prove the absence of any proteolytic enzyme system in the strains. The spectrophotometric enzyme test proved that all lactic acid bacteria from the sourdough process were able to break down gluten. The test was suitable to identify strains with the highest activities as well as strains that were not able to break down gluten. The rapid enzyme test would be suitable for a broad screening of starter organisms for sourdough. Further tests will establish connection between results from the enzyme test and the performance of these strains in dough making and baking.

Acknowledgements This research was part funded by grant aid under the food sub-programme of the operational programme for industrial development, administered by the Department of Agriculture, Food and Forestry and was supported by national and EU funds. We would like to thank the company Böcker for the supply of the sourdough lactic acid bacteria.

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