Field of the invention

The present invention relates to the preparation of bread and other bakery products, in particular products leavened with Saccharomyces cerevisiae.

Background of the invention

Microorganism growth on bakery products may both impair organoleptic properties and be a risk for health due to toxins production. In particular, one of the most frequent problems is flour contamination by sporigen bacteria of the Bacillus genus (B. subtilis, B. cereus, B. licheniformis, B. clausii e B. firmus). The spores of these bacteria survive cooking and cause the so-called "ropy bread" alteration, which consists in progressive softening and formation of threads in the soft part, due to the microorganisms proteolitic activity. Complete and irreversible alteration becomes apparent after 24-48 h storage at 25-30°C; in fact the spores, spread out in the soft part, cause an alteration that cannot be seen from the outside and that makes the product not edible. Therefore, the product is definitely altered before hardening and also before the possible growth of other contaminants (for example fungi and yeasts). On the contrary, fungi and/or yeasts contamination appears on the product surface after 4-5 days storage at room temperature (about 25°C) and accompanies in general the hardening process, while the presence of moulds does not cause alteration of the whole product, but only of some parts that can be physically removed. The ropy bread phenomenon occurs mainly in hot countries, such as those of the Mediterranean basin, but also in industrialised countries such as Australia and South Africa, where the climate is hot and humid and the temperature is around 25-30°C (Pepe O. et al., Appl. Environ. Microbiol., 2003, 69: 2321-2329). Various authors have also reported cases of ropy-bread poisoning, whose symptoms are vomit, nausea, diarrhoea, headache (Voysey, J. Appl.

Bacteriol., 1989, 67: 25-26; Kirschner and von Holy, South African J. ScL, 1989, 85: 425-427). Ropy bread is an actual problem for the bakery industry, since degradation problems due to Bacillus spores in raw materials have recently been reported. In particular, concentrations of Bacillus subtilis and B. lichenifortnis higher than 10 ⁵ cfu/g in the finished product can be a risk for consumers' health (Katina K. et ah, Lebenm.Wiss. u. technol., 35, 2002, pp. 38-45), especially when the product's alteration is not evident. A further predisposition factor to "ropy bread" is the height of the finished product; in fact high breads (for instance sliced bread) promote spore activation and survival in the soft part, where the temperature reached during the cooking process is lower than outside.

Chemical preservatives, such as propionic acid, sorbic acid, acetic acid and ethanol, currently used to prevent microbial contamination in bakery products, may cause some drawbacks. For example, sorbic acid inhibits not only contamination from moulds and bacteria, but also from yeasts, thus causing volume reduction of bread fermented with Saccharomyces cerevisiae, while the use of propionic acid is currently under dispute due to toxicity evidences. Instead, the use of acetic acid as preservative is limited by the unpleasant smell imparted to food. Moreover, since the antimicrobial activity of these compounds directly depends on the amount of undissociated acid and therefore on the food pH, they are not suitable for products with a pH higher than 5 (Spicher G. in: Reed G. (ed.), Biotechnology, vol 5, Verlag chemie, Weinheim, Germany, 1983, p:l-80; Gould G.W. J. Food Microbiol. 1996, 33:51-64, Pepe O. et al, Appl. Environ. Microbiol., 2003, 69: 2321-2329). It should also be noted that, the current European directive on preservatives (95/2/CE) has ordered a reduction in the concentration of propionate and sorbate salts (presently of 3000 and 2000 mg/kg of finished product, respectively), but it has been demonstrated that the use of sub-optimal concentrations of preservatives causes a higher risk of contamination and

alteration. Finally, besides the fact that ethanol does not have a wide action spectrum, it requires quite sophisticated technologies, such as release systems, as an alternative to its distribution on the surface of the finished product just before packaging, which is scarcely effective.

The search for new and natural antimicrobial systems to prevent food contamination is necessary for the food industry in view of the increasing consumers' request for high quality and low-processed food. In this respect, the use of sour dough consisting of lactic bacteria and yeasts to improve storage, rheology, and aroma of several products, among them bread and bakery products, is known. However, sour dough is scarcely used in the industrial production of bakery products, due to long fermentation times and difficulties in handling sour dough. Bread leavened with natural yeast has a more homogeneous and smaller porosity and, as a consequence, retains more gases. Such improved rheological features are due to lower pH values, which regulate amylase activity, thus improving the pastry dough's consistency, and promotes the flour's protease activity, which is activated at pH values of about 4 (De Angelis M. et al., Int. J. Food Microbiol., 2003, 87: 259-270). Bakery products are obtained on an industrial scale through fermentation of Saccharomyces cerevisiae only, due to the fact that this yeast allows rapid leavening and is convenient to use, since it can be purchased as concentrated culture and added when necessary. Bread dough leavened with S. cerevisiae has a pH value not lower than 5.3-5.5.

The quality of leavened bakery products depends also on the bioavailability of the flours' mineral fraction, which is limited by the presence of phytic acid. Flours endogenous phytases hydrolyse phytic acid and increase the bioavailability of the mineral fraction. The activity of endogenous phytases increases as pH approaches 4.

Therefore, it would be advantageous to provide methods for producing leavened bakery products that, although comprising the use of S. cerevisiae,

allow to maintain the pH at lower values (between 4.5 and 4.7), thus preventing the product from becoming ropy, improving its rheological properties and increasing the bioavailability of the flour's mineral fraction, without visible changes.

Lavermicocca P. et al. (Appl. and Env. Microbiology, Sept. 2000,pp. 4084-4090) disclosed the antifungal effect of phenyl-lactic and p-hydroxy- phenyl-lactic acid produced by Lactobacilli added to some kinds of bread in combination with S. cerevisiae as leavening agents.

Detailed description of the invention

It has now been found that, by adding a fermentation product from lactic acid bacteria which contains phenyl-lactic acid or by adding phenyl- lactic acid to the pastry dough, germination of Bacillus spores can be prevented, thus reducing the occurrence of the "ropy bread" phenomenon in bread and other bakery products leavened exclusively with S. cerevisiae.

Accordingly, the present invention relates to a process for the preparation of bread and other bakery products comprising the addition of a fermentation product from lactic acid bacteria, preferably Lactobacilli, which contains phenyl-lactic acid, or the addition of phenyl-lactic acid to a mixture of flour and other ingredients containing S. cerevisiae as the sole leavening agent. According to a preferred embodiment, the mixture is added with a fermentation product from L. plantarum filed with the Belgian Coordinated Collections of Microorganisms, BCCM/LMG- Collection, Gent, Belgio, accession number LMG P-22033. For the purposes of the present application, the expression "fermentation product" means the product obtained by culturing this microorganism in a suitable medium. According to a preferred embodiment, the bacterial cells are removed after the fermentation. Preferably, the medium contains either a mixture of aminoacids, nitrogen bases, salts, sugars and α-ketoglutaric acid, or wheat flour hydrolysate optionally containing a mixture of aminoacids and α-ketoglutaric acid or MRS (Man Rogosa

Sharp). Aminoacids pools suitable for the preparation of the medium are also commercially available. The fermentation product can be added in the lyophilised form, at a concentration (referred to the weight of the lyophilised product) comprised between 0.7% and 0.8% of the flour's weight (1 g lyophilised product/100 g flour); according to a more preferred embodiment, the concentration amounts to 0.75%. As an alternative, the lyophilised product can also be added after dissolution in water. A further embodiment of the invention consists in the addition of phenyl-lactic acid in an aqueous solution at a concentration comprised between 0.15% and 0.22% of the flour's weight (1 g phenyl-lactic acid/100 g flour); according to a more preferred embodiment, the concentration is 0.2%.

According to a particularly preferred embodiment, the process comprises the following steps:

- a first kneading phase wherein the ingredients are mixed;

- a first leavening phase;

- a second kneading phase;

- a second leavening phase;

- rolling out the dough;

- a further leavening phase; and

- baking.

In the first kneading phase all ingredients are mixed, including yeast and either the fermentation product or phenyl-lactic acid. The concentration of S. cerevisiae is higher than I xIO ⁸ cfu/g and is preferably comprised between I x IO ⁸ and IxIO ⁹ cfu/g of pastry dough. The yeast cell suspension is prepared by adding 1O g yeast to 110 ml water. Further to the basic ingredients of the preparation, the pastry dough can be added with spices and/or sunflower seeds, poppy seeds, pumpkin, raisins, olives, etc.

The present invention can be applied in general to leavened bakery products such as croissants, whole-corn breads, special breads, etc.

The invention will be hereinafter illustrated in greater detail in the following experimental section.

EXPERIMENTAL SECTION

EXAMPLE 1 - Process for the preparation of a fermentation product from a L. plantarum LMG P-22033 culture to be added to the dough

To prepare the fermentation product, L. plantarum LMG P-22033 is cultured in a substrate medium (SCl) with the following composition:

ative, the substrate can have the following composition (SC2):

For the preparation of SCl and SC2 all the ingredients are dissolved under stirring at about 50°C. The substrate is sterilised by filtration (0.45 μm filters); pH: 5.6+ 0.2.

100 ml of substrate are inoculated with 2 ml of a bacterial suspension (from a 24 h culture in MRS - Man Rogosa Sharp, Difco at 30°C ) and incubated for 72 h at 30°C. After the growth, the bacterial cells can be removed by

centrifugation (4000 rpm x 10 min., 4°C) and the supernatant sterile-filtered (0.45 filers μm Millipore, Bedford, MA), otherwise the fermentation product is used as such. The resulting fermentation product (pH of about 3.7) (either lyophilised or not) is added to the pastry dough with all the other ingredients. As an alternative, the bacterium can be cultured in Man Rogosa Sharp (MRS, Difco Laboratories), or Potato Dextrose Broth (PDB, Difco Laboratories), or in wheat flour hydrolysate containing the aminoacid pool relevant to the medium SCl and α-ketoglutaric acid. Wheat flour hydrolysate can be obtained by suspending 100 g flour in 500 ml tap water: the resulting suspension is incubated at 30°C for 18 hours under stirring (150 rpm) and then centrifuged at 9000 rpm, 4°C for 15 min. The supernatant is sterile-filtered and added with glucose, fructose and maltose at a final concentration of 1% (w/v). The wheat flour hydrolysed containing the aminoacid pool and α-ketoglutaric acid is obtained by suspending 100 g flour in 500 ml tap water; the resulting suspension is incubated at 30°C for 18 h under stirring (150 rpm) and then centrifuged at 9000 rpm, 4°C for 15 min. The supernatant is filtered through a paper filter and added with glucose, fructose and maltose at a final concentration of 1% (w/v), the aminoacid pool at a final concentration of 3 g/1 and α-ketoglutaric acid at a final concentration of 1 g/1. The substrate is inoculated with the bacterial suspension. In a further embodiment, a substance contained in the fermentation product, i.e. phenyl-lactic acid is added to the pastry dough, together with the other ingredients.

EXAMPLE 2 - In vitro Determination of the Minimal Inhibitory Concentration (MIC) of phenyl-lactic acid against Bacillus spp.

The inhibitory activity of phenyl-lactic acid against three Bacillus species (C3, S6, A3) (Pepe O. et al, Appl. Environ. Microbiol., 2003, 69: 2321-2329) and a B. subtilis ATCC 8473 (American Type Culture Collection) isolated from ropy bread samples was evaluated in vitro in 96 micro-wells plates to determine the MIC. A solution of PLA (10 mg/ml) in phosphate

buffer 0.05 M pH 5.5 is serially diluted to 1 :1 with Isosensitest broth (Difco Laboratories, Detroit, USA) and 100 μl of each dilution is added to the wells in triplicate. 90 μl of Isosensitest broth and 10 μl of inoculum are added to each well. The inoculum of bacterial spores is prepared as follows: cells from a culture grown for 6 days at 30°C on Starch Agar (SA) (Difco Laboratories, Detroit, USA) are resuspended in 0.05 M phosphate buffer at pH 6.5 and heated to 85°C for 15 minutes. The suspension, rapidly cooled in an ice-bath, is centrifuged at 7000 rpm x 10 minutes at room temperature and the spores are collected in 0.05 M phosphate buffer at pH 6.5 and counted with a Thoma cell. 10 μl of a suspension containing 10 ⁴ spores/ml are inoculated in the microplates. The plates are therefore incubated at 30°C for 72 hours and optical density (580 nm) is monitored every 24 h with a spectrophotometer.

Results

Phenyl-lactic acid inhibits all the assayed strains, with MIC comprised between 2500 and 600 μg/ml (Tab. 1). In particular, the spores of the C3 strain are completely inhibited at a PLA concentration of 600 μg/ml (3.75 mM). Phenyl-lactic acid determines 100% inhibition of germination of A3 and ATCC 8473 spores at a 1250 μg/ml (7.5 mM) concentration and the lowest concentration tested (300 μg/ml) causes 50% inhibition of the A3 spores. The S6 strain showed a MIC of 2500 μg/ml (15 mM).

Table 1. Inhibition of Bacillus spp. spores germination by phenyl-lactic acid at pH 5.5

EXAMPLE 3 - Process for the preparation of bread leavened with Saccharomyces cerevisiae only, containing the fermentation product of L. plantarum LMG P-22033 or phenyl-lactic acid and artificially contaminated with bacterial spores before baking

Ingredients:

35O g "00" flour

110 ml water

5 g margarine

5 g sugar

5 g salt

1O g compressed yeast {Saccharomyces cerevisiae)

100 ml water (for the control bread) or the fermentation product of L. plantarum LMG P-22033 (which corresponds to 2.64 g lyophilised product) or water containing phenyl-lactic acid (0.525 g to obtain a final concentration of 0.15% relative to the flour's weight or 0.7 g to obtain a final concentration of 0.2%).

The process is as follows: a) First kneading.

The ingredients (except for the fermentation product of L. plantarum LMG P-22033 or the phenyl-lactic acid solution or the corresponding amount of water) are mixed with the yeast suspension in 110 ml water.

1 ml bacterial spore suspension (5x10 ⁴ - 5><10 ⁶ spores/ml) of a Bacillus strain, prepared as in example 2, is added to the other ingredients so as to obtain a final concentration of about 10 ² - 10 ⁴ spores/g pastry dough, then the fermentation product of L. plantarum LMG P-22033 or the solution of phenyl-lactic acid or the corresponding amount of water is added. b) First leavening (20 minutes). c) Second kneading (15 minutes).

d) Second leavening (20 minutes). e) Rolling out of the pastry dough (30 seconds). f) Last leavening (55 minutes) and measurement of the dough's pH. g) Baking (60 minutes). h) Cooling of the bread in a sterile environment and measurement of the water activity value. i) Weighing of the bread and sealing in polyethylene bags for alimentary use.

1) Storage for 3 or 6 hours at 30°C. m) Homogenization and measurement of the bacterial population (the bread is grinded and mixed in a 1:10 ratio with Bacto Peptone 0.5 g/1. The suspension is left 30 min. at room temperature and homogenised for 2 min. in a Stomacher. 100 μl suspension is plated on Starch Agar plates incubated at 30°C for 48 h).

Results

Bread (500 g) obtained after addition either of the fermentation product of L. plantarum LMG P-22033 or phenyl-lactic acid to the pastry dough has rheological features comparable with those of the control bread, in particular a water activity value of about 0.986 and a height of about 9.5 cm. The pastry dough's pH containing the fermentation product of the bacterium is 4.64, that of the pastry dough with phenyl-lactic acid is 4.7 and that of the control is 5.3-5.5. The addition of the fermentation product of L. plantarum LMG P-22033 to the pastry dough almost completely inhibits germination of B. subtilis ATCC 8473 spores (Tab. 2) (550 cfu/g left over 300 millions cfu/g present in the control bread and 60 cfu/g left over about 100 millions cfu/g present in the control bread) and therefore the fermentation product prevents bread from becoming ropy (Fig. 1 e 2). When the bacterium is cultured in a different medium, such as PDB or flour hydrolysed without the aminoacid

pool and ot-ketoglutaric acid, formation of ropy bread occurs at least 3 days later than in control bread (Fig. 3, 4). When the bacterium is cultured in flour hydrolysed added with the aminoacid pool and α-ketoglutaric acid, an almost complete inhibition of germination of B. subtilis ATCC 8473 spores was observed (2400 cfu/g left over 960 millions cfu/g present in the control bread)(Fig. 5). The addition of phenyl lactic acid to the dough inhibits spore germination of the two Bacillus spp. strains which cause ropy bread (Tab. 2, Fig. 6). Moreover, both the fermentation product and phenyl lactic acid do not inhibit Saccharomyces cerevisiae's growth and therefore do not reduce the bread's volume or modify its aspect.

Table 2 - Inhibition of the germination of Bacillus subtilis ATCC 8473 spores in baking assays with or without the fermentation product of L. plantarum LMG P-22033 cultured in SCl or SC2, after 3 days storage at 30°C

Table 3 - Inhibition of germination of Bacillus spores in baking assays with or without phenyl-lactic acid, after 6 days storage at 30°C

Description of the figures

Figure 1 shows a comparison between bread inoculated with Bacillus subtilis ATCC 8473, added with a fermentation product from Lactobacillus plantarum LMG P-22033 prepared in SCl (A) (no ropy bread) and bread without fermentation product (B) (ropy bread).

Figure 2 shows a comparison between bread inoculated with Bacillus subtilis ATCC 8473, added with a fermentation product from Lactobacillus plantarum LMG P-22033 prepared in SC2 (A) (no ropy bread) and bread without fermentation product (B) (ropy bread).

Figure 3 shows a comparison between bread inoculated with Bacillus subtilis ATCC 8473, added with a fermentation product from Lactobacillus plantarum LMG P-22033 prepared in PDB (A) (no ropy bread) and bread without fermentation product (B) (ropy bread).

Figure 4 shows a comparison between bread inoculated with Bacillus subtilis ATCC 8473, added with a fermentation product from Lactobacillus plantarum LMG P-22033 prepared in the flour hydrolysate (A) (no ropy bread) and bread without fermentation product (B) (ropy bread).

Figure 5 shows a comparison between bread inoculated with Bacillus subtilis ATCC 8473, added with a fermentation product from Lactobacillus plantarum LMG P-22033 prepared in the flour hydrolysed containing the aminoacid pool and α-ketoglutaric acid (A) (no ropy bread) and bread without fermentation product (B) (ropy bred).

Figure 6 shows a comparison between bread inoculated with Bacillus subtilis ATCC 8473, added with a fermentation product from Lactobacillus plantarum LMG P-22033 containing 0.2% phenyl-lactic acid (A) and bread without fermentation product (B).

BELGlAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCM LMG-COLLECTION

Page 1 of Form BCCM/LMG/BP/4/ 03-15A Receipt in the case of an original deposit

Budapest Treaty on the International Recognition of the Deposit of Microorganisms -for the Purposes of Patent Procedure

Receipt- In the case of an original deposit Issued pursuant to Rule 7.1 by the International Depositary Authority BCCM/LMG Identified at the bottom of next page

International Form BCCM/LMG/BP/4/03-154

To: Name of the depositor Paola Lavermicocca

Address Institute of Sciences of Food Production (ISPA)

National Research Council (CNR)

V.1e L. Einaudi 51

1-70125 Bari

Italy

I. identification of the microorganism:

I.1 identification reference given by the depositor:

ITEM21B

I.2 Accession number given by the international Depositary Authority:

LMG P-22033,

II. Scientific description and/or proposed taxonomic designation

The microorganism identified under I above was accompanied by : (mark with a cross the applicable box(es))-. XX a scientific description XX a proposed taxonomic designation

III. Receipt and acceptance

This International Depositary Authority accepts the microorganism Identified under I above, which was received by it on (date of original deposit) :

September 19 , 2003

!V. International Depositary Authority

Belgian Coordinated Collections of Microorganisms (BCCM) Laboratorium voor Microbiologic - Bacteriënversameling (LMG) Universiteit Gent , K.L. Ledeganckstraat 35 B-9000 Gent, Belgium

Signature(s) of person(s) having the power to represent the International Depositary Authority or of authorized official(s):

Dr . D . Janssens, curator IDA

Date : September 29, 2003

BELGlAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCM

LMG-COLLECTION

Page 1 of Form BCCM/LMG/BP/9/ 03-154 Viability statement

Budapest Treaty on the international Recognition .of the Deposit of Microorganisms for the Purposes of Patent Procedure

Viability statement Issued pursuant to Rule 10.2 by the Internationa! Depositary . Authority BCCM/LMG identified on the following page

International Form BCCM/LMG/BP/9/ 03-154

To: Party to whom the viability statement is issued:

Name : Paola Lavermicocca

Address : Institute of Sciences of Food Production (ISPA) National Research Council (CNR) V.1e L. Einaudi 51 I-70125 Bari

Italy 1. Depositor: I.1 Name : Paola Lavermicocca

I.2 Address : Institute of Sciences and. Food Production (ISPA.)

National Research Council (CNR) V.1e L. Einaudi 51 ,

I-70125 Bari Italy II. Identification of the microorganism

11.1 Accession number given by the International Depositary Authority:

LMG P-22033

II.2 Date of the original deposit (or where a new deposit or a transfer has been made, the most recent relevant date) :

September 19 , 2003 III.. Viability statement.

The viability of the microorganism identified under II above was tested on

September 19-22 , 2003

(Give date. In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most recent viability test). On that date, the said microorganism was: (mark the applicable box with a cross) XX viable

!V. Conditions under which the viability test has been performed:

(Fill in if the information has been requested and if \he results of the test were negative).

V . international Depositary Authority

Belgian Coordinated Collections of Microorganisms (BCCM)

Laboratorium voor Microbiology - Bacteriënverzameling (LMG)

Unlversiteit Gent

K.L. Ledeganckstraat 35

B-9000 Gent, Belgium

Signature(s) of person{s) having the power to represent the International Depositary Authority or of authorised official(s) :

Dr. D. Janssens, curator IDA

Date : September 29, 2003

BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS BCCM/LMG-COLLECTIO

Contract BCCM/LMG/DBT1/. n the case of an original deposit or in the case of a new deposit with another International Depositary Authority.

Budapest Treaty of 28 April 1977 on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Contract in the case of an original deposit or in the case of a new deposit with another International Depositary Authority.

Contract BCCM/LMG/DBT1/. number to be filled in by IDA)

Send to:

Belgian Coordinated Collections of Microorganisms (BCCM)

Laboratorium voor Microbiologic - Bacteriënverzameling (LMG)

Universiteit Gent

K.L. Ledeganckstraat 35

B-9000 Gent, Belgium

Between the Belgian Coordinated Collections of Microorganisms - BCCM, represented by:

Laboratorium voor Microbiologie - Bacteriënverzameling (LMG)

Universiteit Gent

K.L. Ledeganckstraat 35

B-9000 Gent, Belgium called hereafter "BCCM/LMG" and the "Depositor" identified at the bottom of this form

It is agreed that if BCCM/LMG accepts the deposit under the Budapest Treaty of the microorganism with the following identification reference, by sending to the depositor the signed form BCCM/LMG/DBT2/. which completes the procedure,

(Indicate here below number, symbols, etc. given to the microorganism by the depositor)

1.The depositor knows and will observe all the provisions of the Budapest Treaty of 28 April 1977 on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations under the Treaty and, more specifically, that he;

a) will provide all necessary information requested by BCCM/LMG, especially the information found on form BCCM/LMG/BP/1 in the case of an original deposit and form BCCM/LMG/BP/3 in the case of a new deposit; b) will pay all necessary fees; c) will not withdraw the deposit for the period specified in Rule 9.1; d) authorizes BCCM/LMG to furnish samples according to the applicable patent requirements; e) will make a new deposit in the event of BCCM/LMG being unable to fαraish samples;

2.The depositor will not hold BCCM/LMG or the Belgian federal office for scientific, technical and cultural affairs responsible for any deterioration of samples during storage when all precautions indicated by him in respect of storage have been taken by BCCM/LMG;

3.The depositor will compensate BCCM/LMG for any damage it may sustain as a consequence of dealing with the organism when all precautions indicated by him in respect of dealing with the organism have been taken by BCCM/LMG;

4.The depositor will indemnify BCCM/LMG against any action brought against it as a consequence of the release of samples, unless such action results from negligence on the part of BCCM/LMG.

Any dispute that may arise shall be governed by Belgian law. Only the courts in the Brussels juridical district shall be competent.

The depositor:

(In the case of a private company, give: full registeied name, legal status and address of the registered office)

Name: Paola Lavermicocca and Francesca Valerio

Address: Institute of Sciences of Food Production (ISPA), CNR, Viale Einaudi 51, 70125 Bari, Italy

Signature: Paola Lavermicocca and Francesca Valerio

Date: 15/09/2003

(Where the signature is required on behalf of a legal entity, the typewritten narαe(s) of the natural ρerson(s) signing on behalf of the legal entity should accompany the signature(s)).

Belgian Coordinated Collections of Microorganisms (BCCM)

Laboratorium vbor Microbiologie - Bacterienverzameling (LMG)

Universiteit Gent

K.L. Ledeganckstraat 35 .

B-9000 Gent, Belgium

Signature(s) of person(s) haying the power to represent the International Depositary Authority or of authorized official(s):