Method for producing a food or a precursor of the same, food or a precursor of the same and a corresponding use

Abstract

A method for producing a food or a precursor of the same, including the steps: (a) providing a mash or a wort or last runnings as a first nutrient medium; and (b) treatment of the first nutrient medium with lactic acid bacteria of the species Lactobacillus rossiae (DSM 15814.sup.T) or with lactic acid bacteria of at least two kinds of species including Lactobacillus rossiae (DSM 15814.sup.T). Furthermore, a corresponding food and corresponding uses are claimed.

Claims

1. A method for producing a food or a precursor of the same, the method comprising: (a) providing a mash or a wort or last runnings as a first nutrient medium; and (b) treating the first nutrient medium with lactic acid bacteria of the species Lactobacillus rossiae (DSM 15814.sup.T) or with lactic acid bacteria at least comprising two species including Lactobacillus rossiae (DSM 15814.sup.T).

2. The method according to claim 1, wherein step (b) includes: treating the first nutrient medium with a mixture comprising lactic acid bacteria of the species Lactobacillus rossiae (DSM 15814.sup.T) and Lactobacillus coryniformis.

3. The method according to claim 2, wherein the species Lactobacillus coryniformis is subspecies Lactobacillus coryniformis subsp. coryniformis (DSMZ No. 20007).

4. The method according to claim 1, wherein the treatment according to step (b) takes place in the presence of a yeast product in the first nutrient medium.

5. The method according to claim 4, wherein the yeast product is selected from the group consisting of an extract, an autolysate, a yeast in fresh form, a yeast in dried form, and combinations thereof.

6. The method according to claim 4, wherein the yeast product is present at a mass concentration in the range of from ≥0.2 to ≤70 g/L, referring to the first nutrient medium.

7. The method according to claim 6, wherein the mass concentration of yeast product is in the range of from ≥8 to ≤50 g/L, referring to the first nutrient medium.

8. The method according to claim 4, wherein at least one of the first nutrient medium and the yeast product is free or substantially free of hop bitter substances, wherein the hop bitter substances include soft resins and hard resins including bitter acids and the known derivatives of these resins and acids; and wherein the first nutrient medium is substantially free of hop bitter substances when a content of hop bitter substances is of at most 15% referring to a content of hop bitter substances which a conventional brewery wort at pitching comprises for a fermentation with a bottom-fermenting or top-fermenting yeast with bitter units in a range of 15 to 38, measured according to an EBC-method; and wherein the yeast product is substantially free of hop bitter substances when a content of hop bitter substances is of at most 20% referring to a content of hop bitter substances which is comprised in a freshly harvested yeast of a first, second, or third generation which was harvested during a fermentation of a conventional brewery wort with bitter units in a range of 15 to 38, measured according to an EBC-method.

9. The method according to claim 4, wherein the yeast product is obtained from a top-fermenting or bottom-fermenting brewery yeast of the genus Saccharomyces.

10. The method according to claim 9, wherein the yeast product is obtained from a top-fermenting or bottom-fermenting brewery yeast of the species Saccharomyces cerevisiae or of the species Saccharomyces carlsbergensis.

11. The method according to claim 9, wherein the yeast is a pure culture yeast or a harvested yeast from the beer production process.

12. The method according to claim 9, wherein hop bitter substances are removed completely or substantially completely from the yeast or the yeast product; wherein the hop bitter substances include soft resins and hard resins including bitter acids and the known derivatives of these resins and acids; and wherein hop bitter substances are substantially completely removed from the yeast or yeast product when a content of hop bitter substances is at most 20% referring to the content of hop bitter substances which is comprised in a freshly harvested yeast of a first, second, or third generation which was harvested during a fermentation of a conventional brewery wort with bitter units in a range of 15 to 38, measured according to an EBC-method.

13. The method according to claim 1, the method further comprising: (c) providing a mash or a wort as a second nutrient medium; and (d) mixing the treated first nutrient medium obtained in step (b) with the second nutrient medium.

14. The method according to claim 13, the method further comprising: (i) processing medium obtained in step (d) to result in a beverage, and/or (k) mixing the medium obtained in step (d) with a beverage.

15. The method according to claim 14, wherein step (i), if present, comprises treating the medium with a yeast of the genus Saccharomyces, and wherein the beverage in step (k), if present, comprises beer.

16. The method according to claim 14, the method further comprising: (t) adjusting a mass fraction of water in the beverage obtained in step (i) or (k), wherein the mass fraction is adjusted to one of: less than 35%, less than 30%, less than 25%, less than 20%, and less than 15%; and to more than 0% or more than 5%.

17. The method according to claim 14, wherein the method further comprises the step: (l) processing the beverage obtained in the step (i) or (k) to result in a non-fluid food, wherein the beverage obtained in step (i) or (k) is mixed with a precursor of the non-fluid food.

18. The method according to claim 17, the method further comprising: (t) adjusting a mass fraction of water in non-fluid food obtained in step (1), wherein the mass fraction is adjusted to one of: less than 35%, less than 30%, less than 25%, less than 20%, and less than 15%; and to more than 0% or more than 5%.

19. The method according to claim 13, the method further comprising: (e) optionally lautering medium obtained in step (d); (f) boiling medium obtained in step (d) or (e) or keeping the medium obtained in step (d) or (e) hot, and optionally hopping of the medium obtained in step (d) or (e); (g) optionally, at least partly removing trub from the medium obtained in step (f); and (h) optionally, setting the temperature of the medium obtained in step (f) or (g) to a pitching temperature.

20. The method according to claim 19, the method further comprising: (i) processing medium obtained in claim 19 to result in a beverage, and/or (k) mixing the medium obtained in claim 19 with a beverage.

21. The method according to claim 20, wherein step (i), if present, comprises treating the medium with a yeast of the genus Saccharomyces, and wherein the beverage in step (k), if present, comprises beer.

22. The method according to claim 20, the method further comprising: (t) adjusting a mass fraction of water in the beverage obtained in step (i) or (k), wherein the mass fraction is adjusted to one of: less than 35%, less than 30%, less than 25%, less than 20%, and less than 15%; and to more than 0% or more than 5%.

23. The method according to claim 20, wherein the method further comprises the step: (l) processing the beverage obtained in the step (i) or (k) to result in a non-fluid food, wherein the beverage obtained in step (i) or (k) is mixed with a precursor of the non-fluid food.

24. The method according to claim 23, the method further comprising: (t) adjusting a mass fraction of water in non-fluid food obtained in step (1), wherein the mass fraction is adjusted to one of: less than 35%, less than 30%, less than 25%, less than 20%, and less than 15%; and to more than 0% or more than 5%.

25. The method according to claim 1, the method further comprising: (e) optionally lautering medium obtained in step (b); (f) boiling of the medium obtained in step (b) or (e) or keeping the medium obtained in step (b) or (e) hot, and optionally hopping of the medium obtained in step (b) or (e), lautered or unlautered; (g) optionally, at least partly removing trub from the medium obtained in step (f); and (h) optionally, setting the temperature of the medium obtained in step (f) or (g) to a pitching temperature.

26. The method according to claim 25, the method further comprising: (i) processing medium obtained in claim 25 to result in a beverage, and/or (k) mixing the medium obtained in claim 25 with a beverage.

27. The method according to claim 26, wherein step (i), if present, comprises treating the medium with a yeast of the genus Saccharomyces, and wherein step (k), if present, comprises mixing the medium with beer.

28. The method according to claim 26, the method further comprising: (t) adjusting a mass fraction of water in the beverage obtained in step (i) or (k), wherein the mass fraction is adjusted to one of: less than 35%, less than 30%, less than 25%, less than 20%, and less than 15%; and to more than 0% or more than 5%.

29. The method according to claim 26, wherein the procedure further comprises: (l) processing the beverage obtained in the step (i) or (k) to result in a non-fluid food, wherein the beverage obtained in step (i) or (k) is mixed with a precursor of the non-fluid food.

30. The method according to claim 29, the method further comprising: (t) adjusting a mass fraction of water in the non-fluid food obtained in step (1) to one of: less than 35%, less than 30%, less than 25%, less than 20%, and less than 15%; and to more than 0% or more than 5%.

31. The method according to claim 1, the method further comprising: (i) processing medium obtained in step (b) to result in a beverage, and/or (k) mixing the medium obtained in step (b) with a beverage.

32. The method according to claim 31, wherein step (i), if present, comprises treating the medium with a yeast of the genus Saccharomyces, and wherein step (k), if present, comprises mixing the medium with beer.

33. The method according to claim 31, the method further comprising: (t) adjusting a mass fraction of water in the beverage obtained in step (i) or (k), wherein the mass fraction is adjusted to one of: less than 35%, less than 30%, less than 25%, less than 20%, and less than 15%; and to more than 0% or more than 5%.

34. The method according to claim 31, wherein the procedure further comprises: (l) processing the beverage obtained in the step (i) or (k) to result in a non-fluid food, wherein the beverage obtained in step (i) or (k) is mixed with a precursor of the non-fluid food.

35. The method according to claim 34, the method further comprising: (t) adjusting a mass fraction of water in the non-fluid food obtained in step (1) to one of: less than 35%, less than 30%, less than 25%, less than 20%, and less than 15%; and to more than 0% or more than 5%.

36. A method of using lactic acid bacteria for producing a beverage or a precursor of the same, the method comprising: utilizing lactic acid bacteria of the species Lactobacillus rossiae (DSM 15814,) lactic acid bacteria of at least comprising two species including Lactobacillus rossiae (DSM 15814T), or a mixture containing lactic acid bacteria of the species Lactobacillus rossiae (DSM 158141) and Lactobacillus coryniformis to treat a first nutrient medium, wherein the first nutrient medium comprises a mash, a wort, or last running.

37. The method of claim 36, wherein the first nutrient medium optionally further comprises a yeast product, and wherein the yeast product, if present, comprises an extract, an autolysate, a fresh form of a yeast, or a dried form of yeast, or combinations thereof.

38. The method of claim 37, wherein the yeast product, if present, is present in a mass concentration in the range from ≥0.2 to ≤70 g/L, referring to the first nutrient medium.

39. The method of claim 38, wherein the mass concentration of yeast product, if present, is in the range from ≥8 to ≤50 g/L, referring to the first nutrient medium.

40. The method of claim 37, wherein first nutrient medium is free or substantially free from hop bitter substances, the yeast product, if present, is free or substantially free of hop bitter substances, or a combination thereof; wherein the hop bitter substances include soft resins and hard resins including bitter acids and the known derivatives of these resins and acids; and wherein “substantially free of hop bitter substances” referring to the first nutrient medium means a content of hop bitter substances of at most 15% referring to the content of hop bitter substances which a conventional brewery wort at pitching comprises for a fermentation with a bottom-fermenting or top-fermenting yeast with bitter units in the range of 15 to 38, measured according to the EBC-method; and wherein “substantially free of hop bitter substances” referring to the yeast product means a content of hop bitter substances of at most 20% referring to the content of hop bitter substances which is comprised in a freshly harvested yeast of the first, second or third generation which was harvested during a fermentation of a conventional brewery wort with bitter units in the range of 15 to 38, measured according to the EBC-method.

41. The method of claim 36, wherein utilizing comprises: utilizing a mixture of lactic acid bacteria of the species Lactobacillus rossiae (DSM 15814.sup.T) and Lactobacillus coryniformis, and wherein the species Lactobacillus coryniformis is subspecies Lactobacillus coryniformis subsp. coryniformis (DSMZ No. 20007).

Description

EXAMPLES

(1) 1. Removing the Yeast of Retardant, Especially of Hop Bitter Substances by Washing

(2) A top-fermenting or bottom-fermenting pure culture yeast or harvested yeast is suspended with brewing water in the ratio 1:9 (50 g process yeast+400 ml water). The resulting suspension is centrifuged for 5 min at 1000 G. Subsequently, the supernatant is discarded and the yeast sediment is re-suspended in 150 ml brewing water. The two last steps may be repeated two to three times.

(3) After the washing, the yeast exhibits a pure, fresh, fruity smell. The originally existing bitterness is missing. In the microscopic compound the yeast cells seem to be intact. In the case of the bottom-fermenting yeast only few damaged cells are occurring. The washed yeast cells exhibit a homogeneous plasm besides the big, round cell nucleus. No cell wall cracks are observed. After vital stain the cells are still colorless (=alive).

(4) 2. Production of a Yeast Autolysate

(5) The process of the yeast autolysis is can be initiated by cracking the cells. In order to do so, the yeasts are mechanically, thermically or chemically treated. The autolysis proceeds then during an incubation of the yeast for multiple hours or a plurality of days at 40 to 55° C. and at a suitable pH-value, such as, for example, at a pH-value in the range of 5 to 7.

(6) A top-fermenting or bottom-fermenting pure culture yeast or harvested yeast is breeded in cast wort. The yeast exhibits a dry substance fraction of about 16 to 17%. It is harvested freshly and washed according to the procedure described before.

(7) Optionally, the yeast cells may be pretreated for decomposition by one or more of the following procedures: a) wet cell decomposition with high-pressure homogenizer; b) ultrasonic treatment (with and without glass balls); c) vortexing (with and without glass balls); and d) addition of propionic acid.

(8) Details according to the single pretreating processes are explained herein below.

(9) The autolysis of the yeast cells itself then takes places by incubation of the possibly pretreated yeast cells for 24 hours at about 53° C. in the incubator (control amplitude: 50 to 55° C.) with permanent stirring of the batch. As a result a fluid autolysate is produced.

(10) 3. Production of a Yeast Extract (Variant of Autolysate)

(11) The produced fluid autolysate as described before is concentrated by water removal. If required, it may be filtered and released from the substances impairing taste.

(12) The main components of the so gained yeast extract are peptides and amino acids as a result of the protein breakdown as well as purines and pyrimidines which are formed by the enzymatic cleavage of the nucleic acids.

(13) 4. Details for the Pretreatment

(14) a) Wet Cell Decomposition with High-Pressure Homogenizer

(15) The mechanical decomposition of the yeast cells was performed with the high pressure homogenizer PANDA Plus 2000 from the company GEA Niro Soavi Germany. The wet cell decomposition by means of the high pressure homogenizer is the preferred decomposition procedure.

(16) As a consequence of the specific fluid dynamics a stationary statistic vacuum (500 to 1500 bar, and more particularly 800 to 1200 bar) arises in the used homogenizer. Thereby, a formation of bubbles arises both within the yeast cell and at the boundary layer between the yeast cell wall and the surrounding medium (cavitation effect). When the vacuum is released at an expansion valve later, this leads to an implosion of the bubbles. This entails a selective rupture of the cell walls.

(17) For preparing the yeast samples the freshly obtained process yeast is diluted with brewing water (1:2, v/v), carbonic acid is removed on the magnetic stirrer and subsequently washed three times according to the procedure described before.

(18) Each yeast sample passes the decomposition procedure two times. The observation of the so treated cells by the light microscope reveals that the cells do not comprise any protoplast after the application of the homogenizer and only the cell shells remain in the compound.

(19) The so decomposed yeast is incubated subsequently in the incubator for about 24 hours at about 53° C. and is autolyzed by the still intact enzymes. In order to stop the autolysis the batches are mashed for about 30 min.

(20) Alternatively, the following procedures may be applied for cell decomposition:

(21) b) Ultrassonicating (with and without Glass Balls)

(22) The yeast sample is diluted with brewing water (10:90, v/v) and subsequently impinged with ultrasonic for 10 minutes (ultrasonic bath: MERCK eurolab USR 46 H).

(23) For amplification of the mechanic forces small glass balls may be added to the yeast sample during the ultrasonic treatment (Company Prolabo/VWR, diameter of 2.5 to 3.5 mm).

(24) c) Vortexing by a Test Tube Shaker (with and without Glass Balls)

(25) The yeast sample is diluted with brewing water (10:90, v/v) and subsequently shaken or stirred intensively with a test tube shaker for one minute (“vortexing”; Vortex Genie 2-shaker: Bender & Hobein AG, level 8).

(26) For amplification of the mechanic forces small glass balls may be added to the yeast sample during the ultrasonic treatment (Company Prolabo/VWR, diameter of 2.5 to 3.5 mm).

(27) d) Addition of Propionic Acid

(28) Propionic acid is added to the yeast sample so that the proportion of the acid in the mixture is 5 Vol.-%. The batch is shaken by hand and then by means of the vortexer. Subsequently the batch is shaken headlong for 15 minutes (TURBULA-Shaker). The samples are settled for 2 hours at room temperature and are shaken again.

(29) 5. Production of Dried Yeast and Yeast Flakes

(30) The yeast suspension in a dry substance fraction of about 15% is sprayed uniformly on a hot roller (rolling dryer from the company VITAM GmbH, Hameln). The yeast cells crack during contacting the roller surface. Cell wall and cell content dry at the roller and are removed latest after 3 seconds as flakes. 10 L of yeast suspension give about 1.5 kg flakes. Subsequently, the yeast flakes are crushed in a mortar.

(31) All of the yeast products named before and all further commercially available yeast products are checked in view of its content of Vitamin B.sub.12: None of the yeast products comprised a detectable amount of Vitamin B.sub.12.

(32) Production of the Yeast Extract (Variant 1 of Dried Yeast)

(33) 20 g of a dried yeast of the stem W34/70 (Company Fermentis, Marcq en Baroeul/France) are suspended in 280 mL a flash pasteurized wort in a 500 mL bottle. Subsequently, the batch is left for 30 min and then is weakly stirred for 30 min (level 1 magnetic stirrer plate).

(34) The batch as described before is incubated (heat stress) at about 57° C. for about 72 hours. Therein, the bottle is opened a little bit so that formed gas can escape.

(35) The so obtained batch is centrifuged at 4500 G for 5 to 10 min. Subsequently, the supernatant is transferred in a sterile vessel. If the supernatant is still too turbid it is centrifuged again. The so obtained supernatant (ca. 250 mL) is the yeast extract according to this embodiment.

(36) Production of the Yeast Extract (Variant 2 of Dry Yeast)

(37) 33.3 kg of a dried yeast of the stem W34/70 (Company Fermentis, Marcq en Baroeul/France) with a dried substance content of 20% are suspended in 150 L first wort in a container. Subsequently, the batch is left for 30 min.

(38) The first wort used here is a non-hopped first wort with 16.5° Plato which was obtained of a mash with a bulk of at least 50% wheat malt. The first wort is pasteurized at 85° C. for about 10 min and is subsequently cooled to room temperature.

(39) An amount of the first wort (ca. 350 L) produced and pasteurized as described before which was not used for the suspension of the dried yeast is heated in a Braun-fermenter at about 57° C. This heated first wort is added as the produced yeast suspension described before by which a total volume of about 500 L of the mixture results. The fermenter exhibits a rise volume of about 50%. The mixture is incubated or fermented at 57° C. for about 72 hours.

(40) Subsequently, the content of the fermenter is pasteurized at 85° C. for about 10 min and subsequently cooled to about 5° C. Dead yeast cells and other solids are removed from the cooled batch by means of microfiltration or centrifugation by which the yeast extract ready to use results. Subsequently, the yeast extract can be stored till usage in a sterile container at 5° C.

(41) Cultivation of the Lactobacills (Variant 1): Lactobacillus rossiae (DSM 15814)

(42) 500 mL MRS-medium (Company Merck, Darmstadt) were set and were autoclaved at 118° C. for 15 min. A lyophilisate of the lactobacills was suspended in the cooled MRS-medium and was incubated at 30° C. for 2 days. The so obtained batch was stored in the cooling room at 4° C.

(43) For inoculation 7.5 mL of the aforementioned obtained batch are suspended in 500 mL fresh MRS-medium.

(44) Cultivation of the Lactobacills (Variant 2): Lactobacillus coryniformis Subsp. Coryniformis (DSM 20001)

(45) A non-hopped first wort with 16.5° Plato is provided which was gained of a mash with a bulk of at least 50% wheat malt. 17 L of the first wort are filled in a 20 L-Cornelius container respectively, are pasteurized/autoclaved at 101° C. for about 31 min and subsequently cooled to 37° C.

(46) The so obtained medium is inoculated with 255 mL of the Lactobacillus coryniformis culture described above and fermented at 37° C. for 48 hours, by which a Lactobacillus coryniformis culture ready for use is resulting.

(47) Cultivation of the Lactobacills: Lactobacillus rossiae (DSM 15814) (Variant 1)

(48) 500 mL of the MRS-medium (company Merck, Darmstadt) were set and were autoclaved for 15 min at 118° C. After cooling 5 g maltose and 5 g yeast extract (company Merck, Darmstadt) were added. Therein, the vessel is filled brimfully due to the fact that Lactobacillus rossiae is microaerophilic or anaerobic. A lyophilisate of the lactobacills was suspended in the MRS-medium and was incubated for 2 days at 30° C. The so obtained batch was stored in the cooling room at 4° C.

(49) 7.5 mL of the batch obtained before were suspended in 500 mL fresh MRS-medium for inoculation.

(50) Cultivation of the Lactobacills: Lactobacillus rossiae (DSM 15814) (Variant 2)

(51) A non-hopped first wort with 16.5° Plato is provided which was gained of a mash with a bulk of at least 50% wheat malt. 17 L of the first wort are filled in a 20 L-Cornelius container respectively, are pasteurized/autoclaved at 101° C. for about 31 min and subsequently cooled to 30° C.

(52) The so obtained medium is inoculated with 255 mL of the Lactobacillus rossiae culture described above and fermented at 30° C. for 48 hours under anaerobic conditions, by which a Lactobacillus coryniformis culture ready for use is resulting. The anaerobic conditions were adjusted by overlaying the medium with CO.sub.2 gas and fermentation in a closed container.

(53) Production of Sauergut According to Embodiments of the Invention (Variant 1)

(54) In a vessel of 100 mL the following ingredients are filled in brimfully:

(55) 72.4 mL flash pasteurized wort, 3 mL of the Lactobacillus rossiae culture as described above, 3 mL of the Lactobacillus coryniformis subsp. coryniformis culture as described above, and 33.6 mL yeast extract as described above.

(56) This batch is homogenized and incubated at 30° C. for 2 days (step (b) of the method) by which a Sauergut is produced as a precursor of a food according to the invention.

(57) Production of Sauergut According to Embodiments of the Invention (Variant 2)

(58) 775.7 L of a non-hoped first wort with 16.5° Plato which was obtained of a mash with a bulk of at least 50% wheat malt is provided. The first wort is mixed with 360 L of a yeast extract described before (produced according to the aforementioned Variant 2) in a fermenter, pasteurized at 80° C. for about 10 min and subsequently cooled to 37° C. The medium is overlayed for the production of anaerobic conditions with CO.sub.2 gas. The so treated first nutrient medium is inoculated with a respective cultivation Lactobacillus coryniformis subsp. coryniformis (DSM 20001) and/or Lactobacillus rossiae (DSM 15814), or with a combination of Lactobacillus rossiae (DSM 15814) and Lactobacillus paracasei subsp. paracasei (DSM 4905), or of Lactobacillus coryniformis subsp. coryniformis (DSM 20001) and Lactobacillus paracasei subsp. paracasei (DSM 4905, each cultivated according to the Variant 2 described above) in a volume ratio of 1:1. The fermenter content is homogenized and is incubated at 37° C. for 2 days under slight stirring (step (b) of the method).

(59) The fermentation may be stopped if the pH-value reaches a value of 3.7. For a defined stop of the fermentation the fermenter content may be pasteurized at 85° C. for 10 minutes. Furthermore, the lactate produced during the fermentation may be separated partially or completely by microfiltration, dialysis or another suitable separation method, if desired. Hereby, the acid taste of the formed Sauergut is reduced which may be desirable depending on the application. Furthermore, the dead cells are separated. Subsequently, there is the possibility to concentrate the so obtained Sauergut for example to 60° Brix in order to reduce the mass and costs of transportation of the so obtained food or a precursor of the same. The concentration step may be performed by means of a vacuum evaporator or another suitable process engineering.

(60) Results

(61) TABLE-US-00001 TABLE 1 Vitamin B.sub.12 concentration after treatment of first nutrient medium Vitamin B.sub.12 First concen- Batch nutrient tration No. medium Lactobacillus Yeast product [μg/100 mL] A1 wort — — 0.05 (blank) A2 wort L.c. — 0.58 A3 wort L.r. — 0.33 A4 MRS L.r. Yeast extract 1.53 (contained in MRS) A5 wort L.r. Yeast extract of fresh 0.31 UG-yeast A6 wort L.r. + L.c. Yeast extract of fresh 4.52 UG-yeast A7 wort L.r. + L.p. Yeast extract of fresh 0.35 UG-yeast A8 wort L.c. + L.p. Yeast extract of fresh 0.32 UG-yeast L.r. = Lactobacillus rossiae (DSM 15814); L.c. = Lactobacillus coryniformis subsp. coryniformis (DSM 20001); L.p. = Lactobacillus paracasei subsp. paracasei (DSM 4905); A6, A7, A8: volume ratio of the lactobacills: 1:1 at normalized number of cells; UG = bottom fermenting; indicated is the total concentration of Vitamin B.sub.12 in the batch, measured according to r-Biopharm AOAC-Methode Nr. 101002, wherein the bio-availability of the Vitamin B.sub.12 formed was confirmed by means of the ADVIA Centaur VB12-Test.

(62) As can be seen from Table 1 above, by the treatment of the first nutrient medium according to embodiments of the invention with Lactobacillus rossiae an elevated concentration of Vitamin B.sub.12 compared to the check may be obtained (see batches No. A3 and A5 vs. A1). A further increase of the Vitamin B.sub.12 concentration may be achieved by the presence of a yeast extract as it is comprised in the MRS-medium (see batch No. A4). A particular high concentration of Vitamin B.sub.12 is obtained when a combination of Lactobacillus rossiae and Lactobacillus coryniformis is used for the treatment of the nutrient medium (see batch No. A6).

(63) In contrast, by applying a combination of Lactobacillus rossiae and Lactobacillus paracasei a concentration of Vitamin B.sub.12 which is achieved is about as high as in the case of the fermentation with only Lactobacillus rossiae (see batch A7 vs. A5). Also a combination of Lactobacillus coryniformis and Lactobacillus paracasei does not lead to an increase of Vitamin B.sub.12 production compared to the use of only Lactobacillus coryniformis (see batch A8 vs. A2). Hereby, it is clearly demonstrated that very high concentrations of Vitamin B.sub.12 is achieved by the combination of Lactobacillus rossiae and Lactobacillus coryniformis, however not by an arbitrary combination of species of lactobacills, even if these include either Lactobacillus rossiae or Lactobacillus coryniformis.

(64) TABLE-US-00002 TABLE 2 Vitamin B.sub.12 concentration and addition of yeast extract Vitamin B.sub.12 First concen- Batch nutrient tration No. medium Lactobacillus Yeast product [μg/100 mL] B1 wort — — 0.05 (blank) B2 wort L.c. — 0.46 B3 wort L.c. Yeast extract of fresh 1.29 UG-yeast B4 wort L.c. Yeast extract of dried 1.78 UG-yeast B5 wort L.c. + L.r. Yeast extract of fresh 4.24 UG-yeast B6 wort L.c. + L.r. Yeast extract of dried 5.72 UG-yeast L.r. = Lactobacillus rossiae (DSM 15814); L.c. = Lactobacillus coryniformis subsp. coryniformis (DSM 20001); B5, B6: volume ratio of the lactobacills: 1:1 at a normalized number of cells; UG = bottom fermenting; indicated is the total concentration of Vitamin B.sub.12 in the batch, measured according to r-Biopharm AOAC-Methode Nr. 101002, wherein the bio-availability of the Vitamin B.sub.12 formed was confirmed by means of the ADVIA Centaur VB12-Test.

(65) As can be seen from Table 2 above, by addition of a yeast extract of dried yeast compared to fresh yeast a further improved yield of Vitamin B.sub.12 may be achieved.

(66) TABLE-US-00003 TABLE 3 Vitamin B.sub.12 concentration and presence of yeast extract Vitamin B.sub.12 First concen- Batch nutrient tration No. medium Lactobacillus Yeast product [μg/100 mL] C1 wort — — 0.05 (blank) C2 wort L.r. — 0.13 C3 wort L.c. + L.r. 0.45 C4 wort L.c. + L.r. Yeast product of 4.93 dried UG-yeast, 50% C5 wort L.c. + L.r. Yeast extract of 4.93 dried UG-yeast, 40% C6 wort L.c. + L.r. Yeast extract of 5.33 dried UG-yeast, 30% L.r. = Lactobacillus rossiae (DSM 15814); L.c. = Lactobacillus coryniformis subsp. coryniformis (DSM 20001); C3 to C6: volume ratio of the lactobacills: 1:1 at a normalized number of cells; UG = bottom fermenting; indicated is the total concentration of Vitamin B.sub.12 in the batch, measured according to r-Biopharm AOAC-Methode Nr. 101002, wherein the bio-availability of the Vitamin B.sub.12 formed was confirmed by means of the ADVIA Centaur VB12-Test.

(67) As can be seen from Table 3 above, this batch also confirms that by the treatment of the wort as the first nutrient medium according to embodiments of the invention with Lactobacillus rossiae an elevated concentration of Vitamin B.sub.12 compared to the blank batch may be obtained. A further significant increase of the Vitamin B.sub.12 concentration may be achieved by the combined treatment of Lactobacillus rossiae and Lactobacillus coryniformis. The yield of Vitamin B.sub.12 further increases significantly in the presence of a yeast extract. Here, a proportion of yeast extract of 50% is not necessary, with a volume fraction of 30% the highest vitamin concentration was achieved in this test series.

(68) Taste testings revealed that the beers produced by means of Sauergut which was produced by the method according to embodiments of the invention exhibited a smell which reminds of bread or sourdough. In particular, this was the case when using yeast extract of fresh yeast. In case of using an addition of dried yeast, the intensity of sourdough bread was significantly less. An analogous impression was observed in view of the taste evaluation of the beers.

(69) In a further batch it should be found out which amount of yeast extract would cause the highest yield of bioavailable Vitamin B.sub.12. For this purpose, the added amounts of yeast extract were varied at same conditions.

(70) TABLE-US-00004 TABLE 4 Vitamin B.sub.12 concentration and amount of yeast product Yeast product Yeast product (vol.-%, referring to (g/L, referring to Vitamin B.sub.12 Batch the first nutrient the volume of the concentration No. medium) first nutrient medium) [μg/100 mL] D1 0 0 0.62 (blank) D2 5 3.8 1.92 D3 10 7.9 2.92 D4 20 17.9 4.48 D5 30 30.6 4.88 D6 40 47.6 3.14 D7 50 71.4 2.46 Used first nutrient medium: wort; used lactobcilli: mixture of Lactobacillus rossiae (DSM 15814) and Lactobacillus coryniformis subsp. coryniformis (DSM 20001); each with the volume ratio 1:1 at a normalized number of cells; yeast product: yeast extract of a bottom fermenting dried yeast, produced by the aforementioned Variant 1; indicated is the total concentration of Vitamin B.sub.12 in the batch, measured according to r-Biopharm AOAC-Methode Nr. 101002, wherein the bio-availability of the Vitamin B.sub.12 formed was confirmed by means of the ADVIA Centaur VB12-Test.

(71) As can be seen from Table 4 above, due to the addition of a yeast product already at an amount of 3.8 g yeast extract to 1 L of the first nutrient medium (corresponds to 5 vol.-% used yeast extract based on the first nutrient medium) an elevated concentration of bioavailable Vitamin B.sub.12 was detected. By an addition of about 30 g yeast extract to 1 L first nutrient medium (corresponding to 30 vol.-% used yeast extract based on the first nutrient medium), the highest concentration with almost 5 μg/100 mL was detected. If the addition of the yeast extract is further increased to about 70 g/L, high concentrations of bioavailable Vitamin B.sub.12 in the nutrient medium are achieved. However, the produced amounts decrease again. According to this batch, an addition amount of yeast product between 4 and 70 g/L, more particularly between 8 and 50 g/L, more particularly between 12 and 40 g/L, and even more particularly between 18 and 35 g/L, referring to the volume of the first nutrient medium seems to result in a particular high yield.

(72) In a further batch it should be found out how the incubation temperature of the yeast affects the production of bioavailable Vitamin B.sub.12 during the production of the yeast product in the production method according to the invention. In order to do so, a yeast extract was produced as a yeast product from bottom fermenting dried yeast according to the Variant 1 described before, wherein the incubation was performed once at 57° C. (standard procedure) and was performed by the variant of 80° C. Additionally, the concentration of the yeast product was varied. All other conditions were the same.

(73) TABLE-US-00005 TABLE 5 Vitamin B.sub.12 concentration and Incubation temperature Yeast product [g/L; referring the Incubation Vitamin B.sub.12 Batch volume of the first temperature concentration No. nutrient medium] [° C.] [μg/100 mL] E1 0 57 0.52 (blank) E2 17.9 57 3.84 E3 35.7 57 5.60 E4 35.7 80 0.20 E5 71.4 57 8.96 E6 71.4 80 0.32 Used first nutrient medium: wort; used lactobacilli: mixture of Lactobacillus rossiae (DSM 15814) and Lactobacillus coryniformis subsp. coryniformis (DSM 20001); each with the volume ratio 1:3 at normalized number of cells; yeast product: yeast extract of bottom fermenting dried yeast, produced by the aforementioned Variant 1 with the incubation temperature 57 or 80° C.; indicated is the total concentration of Vitamin B.sub.12 in the batch, measured according to r-Biopharm AOAC-Methode Nr. 101002, wherein the bio-availability of the Vitamin B.sub.12 formed was confirmed by means of the ADVIA Centaur VB12-Test.

(74) As can be seen from Table 5 above, during this study an increasing concentration of bioavailable Vitamin B.sub.12 was observed with an increasing amount of yeast product (incubation at 57° C.). However, when the incubation temperature was increased from 57° C. to 80° C. during the production of the yeast extract at otherwise same conditions, a significantly lower concentration of Vitamin B.sub.12 resulted.

(75) Variation of the Mixing Ratio of the Lactobacilli

(76) In a further study it was investigated if the yield of Vitamin B.sub.12 may be further increased, if both of the used lactic acid bacteria Lactobacillus rossiae and Lactobacillus coryniformis subsp. coryniformis are used in different mixing ratios.

(77) For all batches 72 mL wort with 33 mL dried yeast and 6 mL suspension of bacteria (in MRS) were mixed wherein each of the suspension of bacteria comprises the following composition:

(78) Batch F2: 1.5 mL L. coryniformis+4.5 mL L. rossiae

(79) Batch F3: 3.0 mL L. coryniformis+3.0 mL L. rossiae

(80) Batch F4: 4.5 mL L. coryniformis+1.5 mL L. rossiae

(81) The respective batch was incubated in the incubator for 48 h at 37° C. Subsequently, the batches were heated in the water bath for 30 min at 95° C. The samples were deep-frozen till the determination of Vitamin B.sub.12.

(82) The contents of Vitamin B.sub.12 were determined microbiologically: 20 mL sample+20 mL (Na-acetate pH 4.5+1% KCN+α-amylase, pepsin).

(83) The further implementation was performed according to the method r-Biopharm AOAC-method No. 101002.

(84) TABLE-US-00006 TABLE 6 Vitamin B.sub.12 concentration and mixing ratio of Lactobacilli Lactobacilli Vitamin B.sub.12 Batch First nutrient (volume ratio concentration No. medium L.c.:L.r.) [μg/100 mL] F1 Wort — 0.05 (blank) F2 Wort 1:3 1.60 F3 Wort 1:1 2.54 F4 Wort 3:1 2.86 Indicated is the total concentration of Vitamin B.sub.12 in the batch, measured according to r-Biopharm AOAC-Methode Nr. 101002, wherein the bio-availability of the Vitamin B.sub.12 formed was confirmed by means of the ADVIA Centaur VB12-Test.

(85) As can be seen from Table 6 above, the increase of the Vitamin B.sub.12-synthesis may be achieved for an increasing L. coryniformis proportion in the inoculum. The absolute concentrations are only to a limited extent comparable to the aforementioned experimental results based on the different states of the lactobacills used.

(86) As a consequence, the proportion of L. coryniformis in the used mixture of lactobacills is at least 50 vol.-%, and more particularly 60 to 90 vol.-%.

(87) Comparison to Prior Art

(88) Conventionally, for the acidification of the mash or the wort the species Lactobacillus amylovorus or Lactobacillus amylolyticus are used which proved for many years in the production of the corresponding Sauergut and the mashes and worts acidified with those.

(89) Therefore, these lactic acid species are characterized by a growth dominance in beer wort due to fast growth. Furthermore, they are characterized by high souring due to a high lactate production. This is based on their homo-fermentative metabolism character. These species grow at high temperatures (to 52° C.) so that high reproduction rates may be obtained.

(90) Furthermore, these species are able to ferment dextrin and starch. Further, they produce a high proportion of L(+)-lactate. The named lactic acid bacteria are not harmful to beer due to the fact that they are hop sensitive and cannot grow at temperatures <30° C. which is of significant importance. Among experts, Lactobacillus amylovorus and Lactobacillus amylolyticus are therefore regarded as suitable organisms for acidification due to the fact that they do not form amines (histamine) or other toxins. Furthermore, they do not form diacetyl or other disadvantageous substances for taste and favor of the resulting products. Finally, they are characterized by easy handling in practical application.

(91) On the other hand, the species Lactobacillus rossiae according to embodiments of the invention, which can be used in combination with Lactobacillus coryniformis according to embodiments of the invention, is considered by experts as a beer spoilage organism. This is also valid for the species Lactobacillus coryniformis. Lactobacillus rossiae is known to the person skilled in the brewing art as a slime former. Both species grow in weakly hopped beer and form diacetyl which leads to a disadvantageously taste profile in the resulting food or beverage, in particular, in the beer. Further, they are able to grow at the conventional beer fermenting temperatures, especially for top-fermenting beer, namely in the range of 15 to 48° C. Furthermore, these species exhibit the disadvantage compared to the species Lactobacillus amylovorus and Lactobacillus amylolyticus conventionally used for acidification that this species is optionally hetero-fermentative, meaning their ability to acidify, is reduced compared to the conventionally used species. As a result about the double amount of Sauergut has to be used compared to conventionally used species. Hereby, larger production facilities are required and the costs linked to the acidification increase.

(92) The aforementioned plurality of disadvantages as well as the disadvantages known by the person skilled in the art of the species Lactobacillus rossiae and Lactobacillus coryniformis examined here means a substantial hindrance for the application of these species or subspecies of the same in the beverage and food producing industry, especially in breweries and malteries, until now.