BACKGROUND OF THE INVENTION Sourdough is a liquid, pasty or dry dough mainly consist- ing of water and flour of cereals, which contains more than 500 millions of live lactic acid bacteria per gram flour (dm) (dm stands for dry matter) and as a result of the metabolic activity of said bacteria has a pH-value below 4. 5. The classical way of starting up a sourdough is to mix flour and water in equal amounts and then leave the dough on a warm place, at 25-35°C, for two days. A so called"spontaneous sour"is then obtained by utilizing the lactic acid bacteria which are naturally occurring in the flour. This"spontaneous sour"is then refreshened by adding additional flour and water and after another day the sourdough is fully developed. In this the lactic acid bacteria are in the stationary phase and the pH-value is within the range of 3. 5-4. 5. In bread baking the sourdough is used in an amount of between 5 and 50% (calculated as the amount of soured flour of the total amount of flour) together with the other ingredi- ents of the bread, the larger amount of rye flour in the bread recipe the higher amount of sourdough.

When a spontaneous sourdough is prepared the microflora is dominated by Gram-negative bacteria belonging to the family Enterobcteriaceae at the beginning, but as the fermentation goes on and pH-value decreases the composition is changed in such a way that it will gradually be dominated by Gram-positive lactic acid bacteria. The naturally occuring yeast flora also increases during the fermentation process. In a mature sourdough the number of lactic acid bacteria usually is about 1-109 CFU/g dough and the number of yeast about 1-105 - 5-107 CFU/g dough. The lactic acid bacteria dominating the microflora of the sourdough belong to the genus Lactobacillus, but representatives of the genera Leuconostoc and Pediococcus have also been found. During the fermentation process many different chemical compounds are formed, which directly or indirectly contribute to the taste of the sourdough and the bread, among which above all lactic acid and acetic acid are of great importance.

In order not to have to repeat the time consuming and often uncertain process which has been described above every time, part of the mature sourdough can be saved and refreshened by adding more flour and water. In this case the lactic acid bacteria and the yeast which are present in a large number in the mature sourdough is utilized. There are examples of sour- doughs which have been kept going in this way for more than 100 years. Sourdough has been used for fermenting bread doughs for several thousands of years. In spite of the increased use of yeast, bakers'yeast was introduced in the middle of the 19th century, the use of sourdough has never ceased but in the oppo- site rather increased in use for the last years. This is mainly due to the positive effects of the sourdough on the properties of the bread, such as taste and aroma, nutritional value and shelf life, both as to fresh keeping properties and resistance against mould and rope formers (Bacillus spp.).

To improve the security in the preparation of sourdough a so called starter culture can be used containing one or more well defined microorganisms in a certain precise amount and having a stated activity. These starter cultures exist in liquid or freezed dried form. In preparing a sourdough by means of a starter culture the latter is mixed with flour and water and left to ferment for up to one day at room temperature or rather at an elevated, for the starter culture optimal temperature between 30 and 37°C. By using a starter culture there will be larger possibilities to get a product of a higher and more constant quality owing to the improved possibilities to control and monitor the sourdough process. By choosing a suitable starter culture the sourdough can also be given certain specific properties.

To overcome the disadvantages which are a consequence of using a sourdough which needs planning 24 hours before the very baking of the bread a number of so called sourdough products have been developed, which can be mixed directly into the bread dough. Said products have a varying composition. In certain cases they consist of an ordinary sourdough which has been dried to a powder and in other cases of a mixture of synthetically produced acids in a flour fraction. The sourdough products which are on the market today are all, however, characterized by containing very low amounts or no amounts at all of the bacteria active at the fermentation in live form. This means that in using said products in bread baking the aroma development or the contribution to the bread fermentation which is obtained with living sourdough is not obtained.

Another way to prepare a sourdough product having a high number of live lactic acid bacteria and optionally yeast and a long shelf life is by freeze-drying. This, however, involves significant costs and therefore has not resulted in any success- ful commercial products.

EP 0 806 144 A2 describes the production of liquid or pasty baking agent which contains live lactic acid bacteria and which can be used either as a starter culture for preparing a sourdough or as a so called pre-dough which is added to the bread dough in an amount of 1-10%. For preparing the baking agent, flour and water are mixed in a ratio of 1 : 9 together with at least one amylase and at least one amyloglucosidase and heated first for 20 minutes to 75°C, then cooled to 55°C and then at least 2 proteases were added and the temperature maintained for 2 hours. The mixture is then sterilized and inoculated with one or more species of lactic acid bacteria. The product ob- tained is said to have a shelf life of up to 6 weeks at cold storage.

The problem thus is to produce a sourdough product at a reasonable cost, which can be stored for a sufficiently long time to allow distribution on the market, but which still main- tains a high content of live bacteria. It is also essential that this sourdough product has a liquid consistancy to facilitate the handling thereof. Consequently, there is still a need of a liquid sourdough product adapted to the market, having an im- proved shelf life and other storage properties.

DESCRIPTION OF THE INVENTION The present invention refers to a process for the prepa- ration of a live, liquid sourdough product with long shelf life.

The process of the invention for the preparation of a live, liquid sourdough product with long shelf life and good storage properties is characterized by the following steps : - flour and water are mixed in a ratio of 1 : 1. 5 to 1 : 3 by weight and 0. 5-4% by weight of salt and 0. 5-5% by weight of calcium carbonate calculated on the amount of flour are added, as well as the enzymes amyloglucosidase and a-amylase, - the mixture is heated to 50-60°C and kept at this temperature for 30-120 minutes, - the mixture is then heated to 90-92°C and kept at this temperature for 15-60 minutes, - the mixture is cooled to 28-40°C, and then - the mixture is inoculated with a starter culture of one or several strains of a facultatively heterofermentative or obligately homofermentative species of Lactobacillus in an amount corresponding to 1.107 - 1. 10 CFU/g flour (dm), - the inoculated mixture is incubated at 28-40°C for 12-24 hours to a pH < 4. 0 and is then cooled to storage tempera- ture, wherein the amount of the added enzymes is chosen to give a sufficiently low viscosity of the obtained final product.

A sourdough product which has been obtained by this process has, when freshly prepared, a pH < 4. 0 and a viable count of > 5.109 CFU/g flour (dm). After cold storage for 10 weeks the pH is still < 4. 0 and the viable count > 5.108 CFU/g flour (dm). The number of live yeast is all the time < 10 CFU/g flour (dm). The invention also refers to the sourdough product prepared by the above process.

The heating to 50-60°C can be compared to a mild scalding process in which the starch is gelatinized and partially de- graded by means of the added enzymes and the enzymes in the flour. By this substances are formed which can subsequently be used by the lactic acid bacteria during the fermentation, but which also directly or indirectly have an influence on the aroma of the bread. In addition a reduction of the viscosity is ob- tained. Depending on the amount of added enzymes and the dura- tion time at 50-60°C the obtained viscosity of the final product can be controlled.

The heating to 90-95°C brings about a killing of bacte- ria, yeast and mould in the mixture. The starch will also be completely gelatinized. A holding time at 90-95°C for a shorter period of time than a quarter of an hour is not sufficient to make the mixture"sterile", but too long a holding time at this temperature will bring about the decomposition of nutrients and aromas.

The flour which is used for the preparation of the sourdough product according to the invention can derive from cereals, such as wheat, rye or barley and should have an extraction rate of 70-100%. The extraction rate and the ash content of the flour are of great importance for the properties of the sourdough. The more of the external hull parts of the cereal grain in the flour, the higher the extraction rate and ash content. As the buffering capacity of the flour to a large extent is located in the external hull layers this means that the acid content of the sourdough will increase with the extraction rate and the ash content. Also from a nutritional point of view it is desirable to have as high an extraction rate as possible, as important nutrient components such as vitamins and fibers are present in a higher concentration in the external hull parts than in the internal parts of the cereal grain. Rye flour is in addition characterized by the falling number, which is a measure of the enzyme content of the flour ; a low falling number stands for a high enzyme content. Rye flour and barley flour are both whole grain flours which are characterized by good nutritional values. However, it could also be desirable to use a wheat flour, especially when the final bread product is a white bread and the colour of the crumb is of importance.

Calcium carbonate is used as a buffering agent having an effect on the fermentation of the sourdough in many ways. Owing to the buffering effect the pH-value will decrease slower in a dough containing calcium carbonate, which means that lactic acid bacteria can grow for a longer period of time giving the final product a higher viable count. Rye or wheat bran also have a buffering effect, but the addition thereof has the disadvantage of significantly increasing the viscosity of the sourdough product.

Salt, such as potassium chloride and preferably sodium chloride, has the ability to inhibit many microorganisms and the addition thereof will thus reduce the activity of the sourdough during storage. When the sourdough is then added to the bread dough the amount of salt is decreased per kilo dough and the activity of the lactic acid bacteria in the sourdough increases again.

Enzymes which are of importance for the decomposition of especially the starch in flour, partly to reduce the viscosity thereof and partly to form nutrients which can be fermented by the added lactobacilli are in the first place different amy- lases, especially a-amylase, and amyloglucosidase. Also other enzymes or enzyme mixtures can be useful. The invention also refers to a process in which a protease is added before the heating of the sourdough mixture. As an example of different enzyme mixtures can be mentioned SAN Super (the main component is an amyloglucosidase supplemented by an a-amylase and a prote- ase), Fungamyl BG (an a-amylase from the fungus Aspergillus oryzae) and AMG (an amyloglucosidase from the fungus Aspergillus niger). An enzyme which can supplement Fungamyl and AMG is Glutenas (a protease isolated from a bacterium). Said enzymes can all be obtained from Novo Nordisk A/S, Bagsvaerd, Denmark.

The amount of enzymes which shall be added can be tested by the man skilled in the art by measuring the obtained viscosity of the final product. In addition to the holding time at 50-60°C and the ratio between flour and water it is also affected by the quality of the flour, especially of the content of enzymes in the flour.

The viscosity of the final sourdough product after dilution of 3 parts of specimen with 1 part of water was mea- sured at a temperature of 25-26°C with a Visco 88 equipment (Metrics Analys AB, Solna, Sweden) set on system 3 and speed 3, giving a moment value of 0. 5-9. 5 mNm.

According to a preferred process rye flour and water are mixed in a ratio of 1 : 2 by weight and 1-2% by weight of salt and 1-2% by weight of calcium carbonate calculated on the amount of flour are added, and then the mixture is first heated to 54-56°C and kept at this temperature for about 1 hour and then to 90-92°C and kept at this temperature for about half an hour. By this a sourdough product is obtained having a high flour content and a high viable count.

The starter culture used in the process of the invention consists of lactic acid bacteria, more precisely of one or several strains of a facultatively heterofermentative or obligately homofermentative species of the genus Lactobacillus.

Obligately homofermentative species of Lactobacillus which can be used according to the present invention are for instance L. acidophilus and L. delbrueckii, which ferment hexoses to lactic acid and which do not ferment pentoses.

The invention especially refers to the use of the facultatively heterofermentative species L. casei and L. plantarum, which ferment hexoses to lactic acid and pentoses to lactic acid and acetic acid. This means that in the flour mix- ture according to the invention they will mainly produce lactic acid but also a smaller amount of acetic acid. The content of bacteria in the starter culture can preferably be 1-5101° CFU/ml.

Said bacteria will give the sourdough a mild, acid aroma and a substantial improvement of the baking properties of the rye flour. In addition the shelf life of the bread is improved both as to freshness and resistance to mould.

The invention especially refers to a process for prepar- ing of a sourdough product in which the starter culture consists of the strain Lactobacillus plantarum I-11, which has been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on August 21,1998, and been given the Acces- sion Number DSM 12383. This starter culture can be used in both rye and wheat sourdoughs.

L. plantarum I-11 has been isolated from an Icelandic sourdough on the substrate MRS supplemented with lo yeast ex- tract and 2% maltose. Incubation took place anaerobically at 30°C. In order to characterize the new lactic acid bacteria, its ability to ferment different carbon sources was investigated by means of API 50 trays and API CHL medium (API System, Montalieu Vercieu, France) and in addition the bacterium was tested on RAPD, randomly amplified polymorphic DNA, giving an"artificial finger print"of the genome of the bacterium. The bacterial isolate was subcultured three times before being tested on API and RAPD. It then turned out that the fermentation pattern on API of the strain I-11 differed from that of the type strain for Lactobacillus plantarum, ATCC 14917T, as to four carbon sources, that is L-arabinose, a-methyl-D-mannoside, melezitose and D- turanose. In all cases the type strain ferments said carbon sources but I-11 does not. The RAPD pattern of the new strain also differed from that of the type strain. The bacterium is kept at -80°C in a special freeze medium containing 3. 6 mM K2HP04, 1. 3 mM KH2PO4, 2.0 mM Na-citrate, 1. 0 mM MgSO4 and 12% glycerol.

The invention also refers to the new strain Lactobacillus plantarum I-11, DSM 12383, for use in preparing a sourdough and other food products.

Obligately heterofermentative species of Lactobacillus are not suitable to use in the process of the invention as they form carbon dioxide during the fermentation giving problems during storage.

An adequate temperature for the fermentation of the sourdough is 28-40°C, preferably about 30-35°C. If a lower or higher temperature is used the properties of the sourdough will be changed.

In the mature sourdough the pH-value is about 3. 4-4. 0 and the number of live bacteria is > 5.108 CFU/g flour (dm). The acid equivalent should be > 20 and < 27 ml 0. 1 N NaOH/5 g flour, that is per amount of sourdough containing 5 g flour. The acid number is a measure of the total amount of acids in a specimen. The analysis is a titration procedure wherein the sourdough specimen is suspended in distilled water and then titrated to pH 8. 5. The amount of 0. 1 N NaOH in ml which is required is the acid equivalent. It is dersirable that the acid equivalent remains relatively constant during storage.

The invention especially refers to a sourdough product which has been prepared according to the invention and which has a viable count of at least 5-108 CFU/g after cold storage for 10 weeks.

In the baking of bread with a sourdough product according to the invention, this is mixed in an amount of about 5-30% (calculated as amount soured flour of the total amount of flour) with the other dough ingredients, such as flour, water, yeast, salt and optional spices. The bread dough is then allowed to ferment for a period of time of one or a couple of hours where- upon the bread is baked. The longer the resting and proofing time the larger is the effect of the live bacteria.

EXAMPLES ExamplJL. Preparation of a sourdough product Rye flour, water, salt and calcium carbonate are mixed with enzyme according to the following recipe : Rye flour 2 kg Water 4 1 Sodium chloride 20 g Calcium carbaonte 20 g Enzyme 7. 5 ml The rye flour is SP Rag Fint having a protein content of 8. 3%, ash content of 1. 37%, and a falling number of 149 s and a water content of 13. 5% (Lilla Harrie Valskvarn, Lilla Harrie, Sweden).

The enzyme is San Super 240 L (Novo Nordisk A/S, Bagsvaerd, Denmark), mainly containing an amyloglucosidase (activity 240 AGU/ml) (AF 22) ), but also a balanced content of a-amylase and protease.

The mixture is heated with a holding time of 60 minutes at 55°C and of 30 minutes at 90°C. The mixture is then cooled to 38°C before being inoculated with the starter culture Lactobacil- lus plantarum I-11 in an amount of 3. 10' CFU/g flour (dm). After the inoculation incubation takes place at 30°C for 24 hours. pH, which should be < 4. 0, and the acid equivalent, which was > 20 ml 0. 1 N NaOH/5 g flour, were checked. The product was cooled and packed before finally being stored at +4°C.

The following properties were measured for the freshly prepared product : lactate content 1. 6 g/100 g sourdough ; acetate content 0. 06 g/100 g sourdough ; ethanol content 0 g/100 g sour- dough. The number of live I-11 bacteria was 1.101° CFU/g flour (dm) and the number of live yeast < 10 CFU/g flour (dm). The viscosity of a sourdough specimen at a dilution of 3 parts of specimen with 1 part water was 0. 51 Pas on a Visco 88 apparatus at 26°C set on system 3 and a rate of 3.

ExampLe2. Preparation of a sourdough product If in the above Example 1 the stated amounts of sodium chloride and calcium carbonate are exchanged for 60 g sodium chloride and 40 g calcium carbonate and the fermentation takes place at 30°C for only 20 hours a sourdough product is obtained having the following properties as freshly prepared : pH = 4. 0 ; acid equivalent 18 ml 0. 1 N NaOH/5 g flour ; lactate content 1. 5 g/100 g sourdough ; acetate content 0. 03 g/100 g sourdough ; ethanol content 0 g/100 g sourdough. The number of live I-11 bacteria was 2.101° CFU/g flour (dm) and the number of live yeast < 10 CFU/g flour (dm). The viscosity of a sourdough specimen at a dilution of 3 parts of specimen and 1 part of water was 0. 52 Pas, measured in accordance with Example 1.

ExampleJL. Preparation of a sourdough product without salt (Comparative example) If the process of Example 2 is repeated with the difference that sodium chloride is not added a sourdough product is ob- tained which as freshly prepared has the following properties : pH = 4. 0 ; acid equivalent 20 ml 0. 1 N NaOH/5 g flour ; lactate content 1. 6 g/100 g sourdough ; acetate content 0. 03 g/100 g sourdough ; ethanol content 0 g/100 g sourdough. The number of live I-11 bacteria was 1.101° CFU/g flour (dm) and the number of live yeast < 10 CFU/g flour (dm). The viscosity of a sourdough specimen at a dilution of 3 parts of specimen with 1 part of water was 0. 52 Pas, measured in accordance with Example 1.

Example A-. Storage test In order to judge the shelf life of the sourdough products which have been obtained in accordance with Examples 1-3 above and which have been cold storaged at a temperature of +8°C, pH, acid equivalent, number of live lactic acid bacteria and yeast, as well as the activity after up to 15 weeks were measured. The number of lactic acid bacteria, MSB, was analyzed on the sub- strate MRS-agar (Oxoid Limited, Basingstoke, England). The incubation took place at 30°C, anaerobically, for 3 days. The number of yeast was analyzed on Potato Dextrose Agar (Oxoid Limited) wherein the pH-value was adjusted to 3. 5 with tartaric acid. The incubation took place at 25°C, aerobically, for 3 days.

The number of live bacteria/yeast is stated in the unit colony forming units (CFU) per gram specimen. Not in any specimen the number of yeast was greater than 10 CFU/g flour. The activity, being a measure of the souring activity of the sourdough, was analyzed by the following test : 45 g sourdough specimen was mixed with 195 g water and 135 g fine rye flour (SP Rag Pint, Lilla Harrie Valskvarn). The sourdough fermentation is then allowed to take place at 30°C and the pH-value of the dough is measured continuously. The time required for the dough to reach pH 4. 0 is measured and is stated in tenths of an hour. In order for the souring activity of the sourdough to be regarded to be sufficient, the time required for the pH-value to reach 4. 0 should be shorter than 10 hours.

The results are given in the following Table.

Table. Result after storage Storage time (weeks) pH Acid equiv. MSB CFU/g Activity h ml/5 g flour to pH 4. 0 flour Sourdough of Ex. 1 0 w 3. 7 20 1.101° 4.1 3 w 3. 7 23 4. 109 - 7 w 3. 7 25 1.109 7.3 10 w 3. 7 25 5 108 8.3 15 w 3. 6 26 3 108 10.6 Sourdough of Ex. 2 0 w 4. 0 18 2.1010 - 5 w 3. 9 20 1.109 7.0 14 w 3. 8 25 6.108 8.5 Sourdough of Ex. 3 0 w 4. 0 20 1.1010 - 5 w 3. 9 25 1.109 8.6 14 w 3. 9 26 7. 10' 17 It is obvious from said tests that the viable count in a sourdough which has been prepared without the addition of salt decreases faster bringing about a deteriorated shelf life. In addition Examples 1-3 show that an addition of calcium carbonate will give a higher number of live lactic acid bacteria in a freshly produced sourdough compared to the number of bacteria in a sourdough without addition of calcium carbonate, which in general does not exceed 5.109 CFU/g flour (dm).

Example 5. Baking of whole bread A coarse, unsweetened but still light whole bread corre- sponding to a dough size of 1 litre is obtained with the follow- ing recipe : Water 2 dl Yeast 40 g Salt 1 tablespoon Sourdough 12 dl Rye flour 16 dl Wheat flour 12-16 dl After fermentation for 10 minutes in a bowl the dough is put on the baking table and rolled into two pieces of the same size which are rolled up and formed into two loaves. They are left to ferment for 60 minutes and then baked at an oven temperature of 225°C for 5 minutes and then at 200°C for about 50 minutes.

ExampLe_6- Baking of white bread, Ciabatta The following ingrediens were mixed to a dough size of 1 liter : Water 10 dl Yeast (sweet doughs) 40 g White syrup 1 tablespoon Salt 1. 5 tablespoon Olive oil 4 teaspoons Sourdough 2 dl Wheat flour 25 dl Work the dough minimally and only to a smooth consistency. Then ferment for 60 minutes and stir some turns. Ferment for another 60 minutes and then pour out the rather sticky dough on a baking table with plenty of flour and roll to a height of 1 cm. Cut squares of 15 x 7 cm and ferment on the plate for 30 minutes.

Bake subsequently at a temperature of 250°C for 12-15 minutes.