DE4025305C2 - - Google Patents

Description

The invention relates to certain heterofermentative lactobacilli and their use as components of a starter culture for the production of a sourdough (cf the claims).

For the production of a suitable sourdough from rye flour, different processes have been developed where the dough in several or just in one step is introduced. The multi-level tours take into account The different temperature requirements of Microorganisms in the dough and control in this way the Sourdough production.

These methods are mainly used to certain ratio of lactic acid to acetic acid, d. H. to create a certain fermentation quotient (cf. Manual sourdough, Behr's ... publishing house, 1987, S 148-161). So z. B. at a temperature of the sourdough of approx. 15 ° C reinforced acetic acid and amplified at about 28 ° C. Lactic acid produced. It is also known the tempera as a function of the electrical conductivity of the  lower the value of the good that has been set, thereby of acetic acid bacteria (DE 37 26 615 A1).

By the duration of the respective temperature levels is Influence on the amount of acid formed. It is that Skilled in the art, in one step at a low level Temperature and a certain known by experience Time to produce a certain amount of acetic acid and in the next stage by a temperature increase during lactic acid production for a certain period of time guarantee.

In addition, in the individual stages in a Überla Were carried out the yeast cell proliferation.

Such methods are reliable, but very time consuming and labor intensive.

As multi-stage methods are known, for example:

• - Foam acid process (5-step process, especially aimed at the propagation of yeasts is);
• - 3-stage acidity:
• a) Basic overnight (classical, empirical developed sourdough guide);
• b) full sour overnight;
• c) Detmolder 3-step duration (the one at a) and b) unavoidable evening work is omitted);
• - Detmolder 2-stage tour:
• a) full-acid maturity 2.5-3.5 hours;
• b) full-sourced 3-4 hours;

A simplification, the so-called Einstufenver drive for the production of sourdoughs. In these become both lactic and acetic acid in one step in a wearable for the bread taste ratio  (Fermentation quotient) produced. This goal is achieved through the use of breeding periods, which according to experience under the conditions of the one-step procedure useful sourdough. The disadvantage is that no purposeful influence on the fermentation quotient can.

A significant yeast multiplication takes place in sourdough not happening. Industrial yeast must be added to the bread dough become.

As a one-step process, for example, it is known:

• - Monheimer Salzsauerverfahren;
• - Detmolder 1-stage guide;
• - Berlin Kurzsauerführung (shortest biological Acidification);
• - Isernhäger Natursauer (salient feature is the deliberate bringing about of hyperacidity of the Sourdough, so the targeted crossing of a by experience determined acid range of the Sourdough, cf. DE-PS 26 11 972).

Instead of breeding periods can be selected for inoculation bacterial starter cultures are used, the konventio nell (by lyophilization or by spray-drying) be prepared (see. EP 01 31 114 A2). Another starter culture is under the Designation Detmold 83 known. This is it a mixed culture of known microorganisms - namely Lactobacillus plantarum, Lactobacillus brevis var. Lindneri and Lactobacillus fructivorans (cf. Paperback for the "Application of Starter Cultures", Rudolf Muller & Co., 6301 Pohlheim, 1989, p 2-3, 10-11).

These starter cultures have the disadvantage that the Microorganisms in their re-seed their otherwise advantageous propagation properties, such as education lose a certain fermentation quotient. To  Two times overcrowding is another usefulness no longer guaranteed.

The above methods offer those in sourdough existing microorganisms very different Conditions. Despite the different conditions Lactic acid bacteria and yeasts are the ones in the Batter out dominant groups.

The known heterofermentative lactic acid bacteria produce a C ₂ body which always contains ethanol and acetate. Such known lactic acid bacteria are, for example, Lactobacillus reuteri (DSM 20016), Lactobacillus fermentum (DSM 20052) and Lactobacillus viridescens (DSM 20410).

For the use of lactobacilli in sourdough Position are the type and quantity of the educated by them Products of great importance. Only through the production Rye flour is able to bake a sufficient amount of acid. The taste of a rye flour bread is essentially by the in the course of sourdough fermentation formed lactic acid and acetic acid. It is the Fermentation quotient of particular interest. A high one Acetic acid content of 25% of the total acid leaves the Bread tastes sour, while 85% lactic acid one produced a mild bread flavor.

The invention is generally concerned with the problem of targeted control of sourdough production and the Properties of baked goods made from it. there is especially on the starter cultures, in particular their components, turned off.

It solves this problem by doing this provides suitable microorganisms, namely the heterofermentative lactobacilli DSM 6037 and DSM 6129 (claims 1 and 2).  

Such heterofermentative lactobacilli have hitherto been not known and are characterized by it from that their C₂ product is free of acetate. In addition, these lactobacilli are regularly structured so that whose C₃ product consists essentially of L-lactate, especially less than 5% D-lactate.

The Lactobacillus DSM 6037 is on June 22, 1990 and the Lactobacillus DSM 6129 at the DSM on July 27, 1990 German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, D-3300 Braunschweig, after the Budapest Treaty has been deposited and received the Entry number DSM 6037 or DSM 6129.

The invention is also the use of a heterofermentative Lactobacillus according to patent claim 1 or 2 iVm with an acetate-containing growth additive and / or one or more acetate-producing other Lactobacillus species as components of a Starter culture for the production of a sourdough (Claim 3).

The acetate-containing growth substance and / or the acetate producing heterofermentative lactobacilli the necessary amount of acetate for the growth of the other Lactobacillus component available. In which Acetate-containing growth additive and / or the acetate production Lactobacilli may be based on known substances and / or Lactobacilli are resorted to, for example the heterofermentative Lactobacillus fructivorans.

The starter cultures thus prepared can be used without control mechanisms for a long time for the sourdough production without their acid production changed significantly. They ensure a constant favorable fermentation quotient and easily permit a multiple inoculation. There are bad at least slight changes, which are without influence on the intended use. In addition, attempts by the Applicant have shown that the use of the lactobacilli according to claim 5 results in a leaven from which excellent bakery products can be produced, in particular breads with highest flavors. This is essentially the acetate-free, ie generally only made of ethanol C ₂ - body of the claimed Lactobacillen responsible. About him and the acetate additive (Growth Substance and / or Lactobacillus), the fermentation quotient can be controlled very accurately.

Lactobacillus DSM 6037 has a certain similarity with Lactobacillus reuteri (DSM 20016) or with Lactobacillus fermentum (DSM 20052). He gets taxo as follows rewrite nomically / biochemically:

• 1. gram-positive and catalase-negative;
• 2. Ferments glucose with formation of gas;
• 3. in the API 50 CH test positive reaction with the following sugars: D-fructose, D-glucose, lactose, maltose, melibiose, D-raffinose, sucrose;
• 4. ammonia from arginine;
• 5. The hydrolyzate of the cell walls contains the amino acids Aspartic acid, lysine, glutamic acid and alanine; in two-dimensional thin-layer chromatography the hydrolysates of whole cells was subjected to epsilon Aminosuccinyl-lysine detected;
• 6. no growth under aerobic conditions agar surface;
• 7. good growth on MRS-Boullion ¹ in anaerobic pot with gas generation kit BR38; MRS-Fertigboullion after De Man et al. (1960), manufacturer's information Universal peptone | 10,0 g meat extract 5.0 g yeast extract 5.0 g di-potassium hydrogen phosphate 2.0 g di-Ammonium 2.0 g D - (+) Glucose 20.0 g Tween 80 1.0 g Sodium acetate × 3 H₂O 5.0 g Magnesium sulphate × 7 H₂O 0.1 g Mangansulphat × H₂O 0.05 g additionally: cysteine 0.05 g Water up to 1000 ml pH 6.5 +/- 0.1
• 8. Grows again in anaerobic medium after resting cells on agar in petri dishes for 4 days Were exposed to air;
• 9. Growth not affected by the addition of CO ₂ to the gas space;
• 10. no growth at 15 ° C, growth at 45 ° C;
• 11. weak growth at initial pH 4.0, good Growth at initial pH of 4.5 and above;
• 12. slow growth on micro-inoculum Broth² and weak growth on the non-selective APT medium according to Evans and Niven (1951) ³ ; ²Micro-Inocolum Broth, manufacturer's information Proteose Peptone no. 3 | 5.0 g Bacto Yeast Extract- 20.0 g Bacto dextrose 10.0 g Potassium di-hydrogen phosphate 2.0 g Sorbitan Monooleate Complex 0.1 g additionally: cysteine 0.5 g least. Water up to 1000 ml pH 6.7 ³Non-selective APT medium for Lactobacilli according to Evans and Niven (1951) Tryptone | 10.0 g Yeast Extract 5.0 di-potassium hydrogen phosphate 5.0 g sodium citrate 5.0 g sodium chloride 5.0 g glucose 10.0 g Tween 80 1.0 g Magnesium sulphate × 7 H₂O 0.8 g Manganese chloride × 4 H₂O 0.14 g Iron sulphate × 7 H₂O 0.04 g additionally: cysteine 0.5 g least. Water up to 1000 ml pH 6.7-7.0
• 13. Poor Growth on Acetate-Free MRS Boullion According to De Man et al. (1960) ¹ , changing the morphology of short (cocoa) rods to very long and curved rods;
• 14. Reduction of doubling time t _D = 7.5 h at 0.05 g / l sodium acetate tri-hydrate to t _D = 3.0 h at 2 g / l; no further shortening of the doubling time with increase of the acetate concentration;
• 15. Produces in an MRS modified medium ¹ containing 10 g of glucose (instead of 20 g of glucose) and 0.2 g of sodium acetate trihydrate (instead of 5.0 g of sodium acetate trihydrate) per liter under anaerobic conditions (with oxygen-free Nitrogen gassing, addition of 0.05% cysteine before sterilization) L-lactate and ethanol (D-lactate less than 5% of the total amount of lactate);
• 16. Ferricates sugar via the pentose phosphate route; To detect phosphoketolase activity;
• 17. may lyophili if added stabilizer and then at -20 ° C for at least 3 months are stored, the germ count only insignificant decreases.

The growth and product formation of DSM 6037 under anaerobic conditions with pressure equalization in the medium ⁴ reproduced below shows the attached Fig. 1.

⁴Modified MRS medium for analysis of fermentation products Universal peptone | 10,0 g meat extract 5.0 g yeast extract 5.0 g di-potassium hydrogen phosphate 2.0 g di-Ammonium 2.0 g D - (+) Glucose 10.0 g Tween 80 1.0 g Sodium acetate × 3 H₂O 0.2 g Magnesium sulphate × 7 H₂O 0.1 g Mangansulphat × H₂O 0.05 g additionally: cysteine 0.5 g Water up to 1000 ml pH 6.5 +/- 0.1

The apparent in Fig. 1 small amount of acetate is due to the acetate content of the medium ^{4 used} . The decrease in the acetate content is explained by the acetate consumption of DSM 6037. Furthermore, FIG. 1 shows a two-phase growth of DSM 6037, preference being given to product formation in phase I. By the end of Phase II DSM 6037 consumes 17% of the added acetate. In Phase II, much ethanol and L-lactic acid are formed. The ratio of L-lactic acid to D-lactic acid was 1 / 0.02 at the end, and the ratio of L-lactic acid to ethanol was 1 / 1.2.

Lactobacillus DSM 6129 is as follows to describe taxonomically / biochemically:

He agrees with the aforementioned taxonomic / biochemical Parameters 1 to 17 with DSM 6037 match, but fermented additionally galactose in the API 50 CH test.

DSM 6037 and DSM 6129 were isolated as follows: A sourdough sample was diluted 1:10 with saline and centrifuged. The supernatant was diluted and diluted to MRS Agar streaked. The plates were at 37 ° C in Anaerobic pot with Oxoid gas kit BR 38 two to three days incubated. The results obtained by repeated spreading Pure cultures were identified by the API 50 CH assay. Notwithstanding the manufacturer 's instructions, the Microreaction vessels not sealed with paraffin oil, but the test strips were placed in the anaerobic pot with gas Kit incubated.

The lactobacilli according to the invention can be, for example be brought into a storable form as follows:

400 ml of modified MRS medium (see parameter number 15 above) in 500 ml laboratory bottles with screw cap were aerated with oxygen-free nitrogen before 0.05% cysteine was added and the bottles were closed with a screw cap. The medium was inoculated with 8 ml of a 10 hour old preculture in MRS broth and incubated at 37 ° C. After 16 hours, an optical density OD ₅₇₈ of about 2 and a pH of 4.4 was achieved. The cells were separated from the culture broth by centrifugation, washed with saline and suspended in a 10% skimmed milk solution, frozen and lyophilized.

The lactobacilli according to the invention can be, for example used for sourdough production as follows:

• - 20 liters of water at a temperature of 28 ° C-30 ° C are placed in a commercially available 75 l acid alarm clock bel, mostly a plastic bucket.
• - 30 g of a starter culture are trickled into the water with simultaneous whisking with a whisk. The starter culture is composed as follows: a) Lactobacillus DSM 6037 | 6.0 g b) Lactobacillus fructivorans (acetate producer) 1.2 g c) a yeast strain 2.5 g d) dextrose as a carrier 20.3 g
• - Then 10 kg of rye flour are added and well mixed.
• - The mash thus obtained is at 24 ° C-28 ° C 18-24 Hours left. The quality of the sourdough obtained regularly becomes significant increased, if you do not put the mash in the acidity bucket, but in a well-known Sauerteiganla ge, with or without kneader, stand, d. H. acidify, if necessary let it acidify.

The above embodiment may be changed as follows become:

• - 15 liters of water
• - 15 kg of rye flour
• - 26 ° C-28 ° C
• - the remaining parameters unchanged or
• - 11.25 l of water
• - 18.75 kg of rye flour
• - 26 ° C-28 ° C
• - the remaining parameters unchanged.

Instead of DSM 6037 can also DSM 6129 or a mixture of these Strains are used. As acetate producers can various heterofermentative lactobacilli strains be used.

Claims (3)

1. Heterofermentative Lactobacillus DSM 6037. 2. Heterofermentative Lactobacillus DSM 6129. 3. Use of the lactobacilli according to claim 1 or 2 with an acetate-containing growth additive and / or one or more acetate-producing other Lactobacillus species as components a starter culture for the production of Sourdough.