Abstract:
A culture medium which is completely free of chemical additives and which can be used for the individual culture of yeast and of lactic acid bacteria or for the coculture of yeasts and lactic acid bacteria is prepared. The preparation of the medium is carried out by a process comprising making in a bioreactor a first medium using a dilute aqueous mixture containing a yeast autolysate and whole-meal or wheat germ, starch and gluten. Alpha-amylase enzymes and amyloglucosidase are added thereto for hydrolyzing the starch into a sugar. Furthermore proteolytic enzymes of food quality are also added for hydrolyzing gluten into aromatic peptides and free amino acids for microbial growth. The first medium is further sterilized and table salt may be added, however, no other chemical additives are ever added to the first medium. A second medium is prepared similarly as the first medium, however, in place of the second medium containing gluten it contains proteins for which the proteolytic enzymes of food quality are added for hydrolyzing the proteins into aromatic peptides and into free amino acids for microbial growth. The second medium is also further sterilized and table salt may be added, while no other chemical additives are added; and furthermore, the hydrolyses of the first and second mediums are carried out without pH regulation. The mediums are then combined to form a culture medium useful for making yeast cultures, lactic acid bacteria cultures and cocultures of yeast and lactic acid bacteria.

Description:
This application is a continuation Ser. No. 08/441,245 filed on May 15 1995, now abandoned. 
    
    
     FIELD OF THE INVENTION 
     This invention refers to a process for preparing culture mediums which can be used for the individual culture of yeast and of lactic acid bacteria or for the coculture of yeasts and lactic acid bacteria, as well as to the use of said culture medium. 
     BACKGROUND OF THE INVENTION 
     Generally, baker&#39;s yeast is obtained from a culture medium consisting either of sugar molasses and chemical additives, or of cereals. However, said culture mediums necessitate a separation between the medium and the microorganism after the growth of the latter, in order to obtain a sufficient concentration of microorganisms permitting a panification under conditions which are compatible with an industrial production. 
     In the case of a so called &#34;sour dough&#34; panification, the production of the sponge necessitates successive pre-cultures and a long adaptation of the microorganisms to the cereal environment, which both are incompatible with an industrial production. 
     SUMMARY OF THE INVENTION 
     A first object of the present invention is to eliminate said disadvantages of the prior art. 
     Another object of the present invention is to create culture mediums which can be used for the individual culture of yeast and of lactic acid bacteria or for the coculture of yeasts and lactic acid bacteria, which are free from any chemical additives, and which permit a direct use of the cultivated microorganisms for industrial panification. 
     The foregoing and other objects, advantages and features of the present invention can be attained by a process comprising the following steps: 
     preparing a diluted aqueous mixture comprising at least whole-meal and/or wheat germs as well as yeast autolysate (hereafter called &#34;diluted mixture&#34; for convenience); 
     completely hydrolyzing the starch in this mixture into fermentable sugars by the action of at least one alpha-amylase and of at least one amyloglucosidase; 
     gently hydrolyzing in this mixture at least part of the gluten by means of proteolytic enzymes of food quality into aromatic peptides and into free amino acids, and this in sufficient quantity to assure, when using the culture medium, a maximum microbial growth; and 
     sterilizing the obtained product. 
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Preferably, said diluted mixture contains also table salt, and particularly sea salt. 
     The process according to the present invention, depending on the composition of said diluted mixture, produces two different mediums, one called &#34;base medium&#34; and the other called &#34;culture medium&#34;. 
     Preferably, the proteolytic enzymes comprise at least two different proteases, one being an endoprotease, and the other being an exoprotease. 
     Preferably, the hydrolyses is executed in a bioreactor without pH regulation. 
     The culture mediums obtained according to the present invention are especially useful for the culture of yeasts, in particular those of the type Saccharomyces cerevisiae, preferably isolated from a natural sponge, and more particularly of the strain Saccharomyces cerevisiae steineri DSM 9211. 
     Likewise, the culture mediums obtained according to the present invention are especially useful for the mixed culture and/or the sequential culture of yeasts with lactic acid bacteria, particularly of yeasts of the type Saccharomyces cerevisiae, preferably isolated from a natural sponge, and of lactic acid bacteria of the genera Lactobacillus, Pediococcus and/or Leuconostoc, preferably also isolated from a natural sponge. They particularly allow the culture of the strain Saccharomyces cerevisiae steineri DSM 9211 in a mixed culture and or a sequential culture with one or several of the strains Lactobacillus plantarum DSM 9208, Lactobacillus brevis DSM 9209, Pediococcus pentosaceus DSM 9210 and Leuconostoc mesenteroides DSM 9207. 
     In this case, the yeast strain is discontinuously grown in a bioreactor in a mixed and/or sequential culture alimented with one or several bacterial strains, depending of the nature of the pacification product which is finally to be produced. 
     This permits the control of the metabolism of each microorganism, thus permitting to act on the concentration of the final metabolites, particularly ethanol, lactic acid and acetic acid. The choice of the microorganismes, based on their metabolic particularities, especially on the homofermentative or heterofermentative ways, and particularly the regulation of the depending enzymatic activity of pyruvate oxydase oxygen (cf.: Frey, Le Lait 1992) enables control of the concentration of acetic acid in the final mixture in a very delicate manner. 
     In the coculture process, part of the final metabolites produced by one of the microbial species is reused by the other species, which influences the final organoleptic qualities (reused lactic acid and production of acetic acid). 
     The sequential system permits to eventually delay the bringing into action of one microorganism with respect to another, or others, respectively. 
     The process according to the present invention offers several possibilities for carrying through the mixed and/or sequential culture: 
     (1) To culture simultaneously one or several yeasts and one or several lactic acid bacteria strains (mixed culture). 
     (2) To culture first one or several yeasts for a given period of time, and to culture thereafter, in the obtained yeast culture, one or several lactic acid bacteria strains (sequential culture). 
     The culture of the lactic acid bacteria itself may by simultaneous (i.e. several strains are inoculated at the same time), or may be delay (i.e. a first strain is set in a coculture with the yeast or several yeasts at time t 1 , then a second strain is introduced at time t 2 . . . t x ). 
     In the same way, the culture of the lactic acid bacteria can precede the start of the coculture with the yeast, or several yeasts, respectively. 
     The following Table summarizes these possibilities in a simplified scheme. 
     Abbreviation: L.a.b.=Lactic acid bacteria 
     
                       TABLE______________________________________Culture Types______________________________________(1)  ##STR1##(2)(a)  ##STR2##(b)  ##STR3##(c)  ##STR4##(d)  ##STR5##(e)  ##STR6##______________________________________ 
    
    
    
     In all cases, the use of the said microorganisms, cultivated in the culture medium prepared according to the present invention, allows in the industrial panification the generation of characteristic organoleptic qualities. 
     EXAMPLE 1 
     Ingredients Used for the Preparation of the Culture Mediums 
     The following ingredients are used for the preparation of the culture mediums described hereafter: 
     Wheat kernels, ground before use to safeguard the integrity of the nutritional values. A typical analysis of the product is as follows: 
     water approximately 13%, 
     total proteins approximately 12%, 
     carbon hydrates approximately 69%, 
     total lipids approximately 2%, 
     starch approximately 59%, 
     ash 1.5%. 
     Wheat germs, ground at low speed with controlled reduced heating. A lipid content from 11 to 12% and a starch content of less than 10% should be retained in order to guarantee the quality of the product. A typical analysis of the product is as follows: 
     water approximately 13%, 
     total proteins approximately 31.5%, 
     carbon hydrates approximately 25%, 
     total lipids approximately 11%, 
     starch approximately 8%, 
     ash 5%. 
     Yeast autolysate of food quality providing the medium with vitamins and amino acids. A typical analysis of the product is as follows: 
     water approximately 3.5%, 
     total proteins approximately 50.5%, 
     carbon hydrates approximately 32%, 
     total lipids approximately 5%, 
     starch approximately 1.5%, 
     ash 7.5%. 
     Sea salt 
     Industrial water. 
     EXAMPLE 2 
     Preparation of Milieu Called &#34;Dosage Medium&#34; 
     8 liters of water, 1660 grams of ground wheat kernels, 1000 grams of wheat germs, 100 grams of yeast autolysate, 30 grams of sea salt, and 1 milliliters of an alpha-amylase solution (16 unites RAU/gram of starch to be hydrolyzed) are mixed in a bioreactor of a volume of 15 liters. 
     Thereafter, the mixture is heated to 85° C. for 20 minutes and then cooled to 75° C. Thereafter, 2 milliliters of the same enzyme are added. The temperature is maintained for 20 minutes. 
     The mixture is cooled to 60° C. Then, 50 milliliters of a solution of amyloglucosidase (16,700 AGI/milliliter) are added. The action of the enzyme is maintained for 90 minutes. The mixture is cooled to 50° C. and subjected to the hydrolysis by two specific proteases, the first one being purified and fractionated papaine, and the second one being fractionated pancreatine. 
     1.5 milliliters of the first protease per kilogram of flour and 2.3 grams of the second protease per kilogram of flour are used. The action of the proteases lasts 220 minutes. 
     The obtained culture medium is sterilized at 120° C. for 20 minutes. This medium, which is perfectly stable is stored at +4° C. 
     At no time during the preparation of the medium are any chemical additives involved. The final pH of the medium is about 6.0, for example 5.5 to 6.5. 
     The addition of alpha-amylase in two lots to the medium called &#34;dosage medium&#34; avoids the irreversible gelatinisation of the starch at the moment when the temperature raises above 65° C. 
     EXAMPLE 3 
     Preparation of the Medium Called &#34;Base Medium&#34; 
     This medium serves for the inoculation called in technical terms &#34;tank bottom&#34; in the production system of the biomass according to the process of the discontinued alimented culture (feed batch). 
     10 liters of water, 500 grams of wheat germs, 70 grams of yeast autolysate and 30 grams of sea salt are mixed in a bioreactor. 
     The medium is subject to hydrolysis by alpha-amylase (2 milliliters/kilogram of wheat germs) for 20 minutes at 75° C., thereafter to the action of the above-mentioned two specific proteases, i.e. purified and fractionated papaine and fractionated pancreatine, at 50° C. for 240 minutes. The medium is stored at +4° C. 
     At no time during the preparation of the medium are any chemical additives involved. The final pH of the medium is about 6.0, for example 5.5 to 6.5. 
     EXAMPLE 4 
     Analysis of the Liberated Sugars 
     For analysis, the liberated sugars are measured by high performance liquid chromatography, and the amino acids liberated by the hydrolysis under the action of the protease are measured by means of the ninhydrine reagent (S. Moore and W. H. Stein, J. Biol. Chem. 176, 367, 1948). 
     The obtained average values are as follows: 
     Dosage Medium (Example 2): 
     glucose approximately 86.5 grams/liter, 
     maltose approximately 11 grams/liter, 
     amino acids approximately 9 grams/liter. 
     Base Medium (Example 3): 
     glucose approximately 6 grams/liter, 
     maltose approximately 12.5 grams/liter, 
     amino acids approximately 6.5 grams/liter. 
     EXAMPLE 5 
     Use of the Culture Mediums for the Culture of Yeast 
     5 liters of a base medium, prepared according to Example 3 above, are introduced into a 15 liter bioreactor, having an useful capacity of 10 liters. 
     A strain of yeast identified as Saccharomyces cerevisiae steineri, and deposited with the German Collection of Microorganisms (DMS) under No. 9211, is added to this base medium. The strain had been cultivated on the dosage medium prepared according to Example 2 above. 
     The strain is stored at -80° C. on a cereal base containing glycerol. The strain is reisolted on the solid cereal base, and is kept on the same medium by successive reinoculation every 15 days. 
     An isolated colony is inoculated into the liquid cereal medium. A second culture is made with 20 milliliters of the first one in an Erlenmeyer of 500 milliliters, containing 200 milliliters of the cereal medium. The cellular density obtained after 16 hours of culture under agitation is 3.0.·10 8  cells per milliliter. A third culture is prepared from the second one with 600 milliliters of the previous culture. The cellular density obtained after 8 hours at 30° C. is 2.5·10 8  cells per milliliter. The glucose is entirely metabolized, and the measured ethanol content is about 25 grams/liter. 
     600 milliliters of this yeast culture in its exponential phase are added to said 5 liters of the base medium. The mixture is continuously alimented with dosage medium. The temperature is maintained at 30° C. The pH, which is continuously measured, is not regulated, and the pO 2  is kept above 10%. 
     The alimentation speed is determined by two parameters which are essential for the good course of the process: 
     (a) The Parameter Ethanol Concentration of the Culture Medium: 
     Yeast is a microorganism capable of multiplying in aerobiose and/or in anaerobiose. The aerobic metabolism is favorable to the growth of the biomass, whereas the anaerobic metabolism leads to the production of ethanol and of secondary derivatives which are searched for their organoleptic qualities. The process according to the present invention allows for control of the metabolic flow between these two extreme ways, permitting a good growth and therefore obtaining a final biomass which is capable of being used in the direct panification under &#34;industrial&#34; time conditions. 
     The production of ethanol indicates a metabolic flow into the anaerobic pathway. 
     In the described process, the ethanol concentration is systematically examined and maintained between 0.5 and 10 grams/liter. If the ethanol concentration drops below the limit of 0.5 grams/liter, the alimentation speed of the dosage medium is raised. On the other hand, the alimentation speed of the dosage medium is reduced if the ethanol concentration exceeds the limit of 10 grams/liter. 
     (b) The Aeration of the Culture Medium: 
     This parameter is continuously determined by continuously measuring dissolved oxygen, and is controlled in two ways, i.e. by controlling the entry of air into the bioreactor and/or by diminishing the rotation speed of two agitator blades of the Rushton type (Size of the blades: one half of the diameter of the bioreactor), allowing the control of O 2  the transfer. 
     In the described process, the partial pressure of O 2  is kept above 10%. The agitation speed varies between 500 to 1200 rpm, and the exit of sterile air is from 0 to 30 liters/minute. 
     Under the described conditions, the culture is fed during 16 hours. The total alimentation during this period is 4 liters. The cellular density of the yeast reaches 2·10 9  cells per milliliter (Density at start: 2.5·10 7  cells per milliliter). 
     The obtained culture is rapidly cooled to 3° C. and can then be kept without noticeable loss of its qualities for 21 days. 
     Variant: 
     A postfermentation on flour in the ratio of 50 to 300 grams/liter of ferment for 6 hours at 20° C., followed by cooling to 3° C., allows a storage during 30 days and the obtaining of a panification product having the organoleptic qualities of the &#34;sponge&#34; type. 
     Panification: 
     The liquid ferments (obtained directly from the bioreactor or after postfermentation) are used directly in a concentration of 20% (weight of flour/volume of ferment) for making a traditional dough from flour, water and salt. 
     Variant: 
     The ferments are centrifuged. The reduction of their water content raises their stability and allows to considerably diminish the concentration in the panification process. Thus, a centrifugation at 8,500 g (gravity) makes it possible to obtain a ferment containing 75% of water, which can be used in a concentration of 6 to 8% in the panification mixture. 
     The density of the obtained breads is identical with that of bread obtained by using the traditional pressed yeast (cultivated on sugary molasses) and used in a concentration of 2 to 3% on the weight of flour. 
     EXAMPLES 6 to 8 
     Use of the Culture Mediums for the Mixed Culture of Yeast and Lactic Acid Bacteria 
     Preliminary Remark 
     The ultimate aim is to obtain a preparation composed of a mixed culture allowing the preparation of a bread which is comparable to a bread made with traditional sponge. The mixed microbial growth brings about numerous interactions of the type commensalism, mutualism and ammensalism between the strains involved. The process described hereafter tries to make a compromise in producing Lactobacilli in sufficient quantity to produce flavors and a characteristic acidity in the breads, and to secure a sufficient proportion of yeasts (rising agents) which guarantee a well aerated bread with good density without further addition of yeast. 
     EXAMPLE 6 
     The culture starts with the growth of yeast. 5 liters of the base medium and 600 milliliters of a culture of Saccharomyces cerevisiae in an Erlenmeyer are continuously introduced into a 15 liter bioreactor, having a useful capacity of 10 liter. The mixture is fed with the dosage medium, and the temperature is maintained at 30° C. The pH, which is continuously measured, is not regulated. At the start the pH is 6, and it goes to 4.0 to 5.0 at the end of the mixed culture, depending on the strain of lactic acid bacteria used. The ethanol content is maintained between 0.5 and 10 grams/liter, by submission of the alimentation of the dosage medium. The agitation is effected by means of two agitator blades of the Rushton type (Size of the blades: one half of the diameter of the bioreactor--Transfer coefficient: 600 mmoles O 2  /liter--Agitation speed: 500 to 1,200 rpm--Exit of sterile air varying: from 0 to 30 liters/minute). Said air exit depends on the partial pressure of oxygen which is continuously measured by means of a pO 2  electrode (Ingold), and is kept above 10%. 
     The inoculation of a lactic acid bacteria, i.e. Lactobacillus plantarum, is made after 8 hours and may be used to adjust the concentrations of the various microorganisms and to influence thereupon the organoleptic qualities of the final product. 
     EXAMPLE 7 
     This example shows a coculture which consist, at the start, of a mixed culture of yeast and Leuconostoc mesenteroides. This mixed culture is followed for 8 hours. After these 8 hours a pre-culture of Lactobacillus plantarum is added. The final product is obtained after 18 hours. 
     EXAMPLE 8 
     This example shows a coculture of the same type as that of Example 7, but in this test Leuconostoc mesenteroides is replaced by Lactobacillus brevis at the start of the coculture. Lactobacillus plantarum is added after 8 hours. 
     It is interesting to note the increase in production of lactic acid as compared with Example 2 (2.40 grams/liter against 1.81 grams/liter). 
     The obtained cellular density ratios between yeasts and bacteria are 3 to 5·10 9  yeast to 3 to 5·10 9  bacteria. 
     The final culture is cooled to 3° C. as quickly as possible. It can be stored for 8 days without loosing its panification capacity. It is used in a concentration of 20% (weight/volume). 
     The taste of the &#34;sour dough&#34; of the breads is excellent. 
     The Leuconostoc mesenteroides strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9207. 
     The complete taxonomic description for the Leuconostoc mesenteroides strain is as follows: 
     Taxomony of Leuconostoc mesenteroides DSM 9207 (Morphological and physiological characteristics) 
     
         ______________________________________Taxomony of Leuconostoc mesenteroides DSM 9207(Morphological and physiological characteristics)______________________________________Characteristics of colony             Diameter of colony:(2 days, MSR Agar)             0.5 to 1 mm, grey, smoothCell form and length             Cocci to short-rod-shaped(MRS Bouillon)Lactic acid configuration             DGrowth at 15° C.             Reaction positiveGrowth at 45° C.             Reaction negativeEnd pH in MRS Bouillon             4.2Gas formation out of             Reaction positiveglucoseAminonia out of arginine             Reaction negativeDiamino pimelic acid             Reaction negative______________________________________ 
    
     The Lactobacillus plantarum strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9208. 
     The complete taxonomic description for the Lactobacillus plantarum strain is as follows: 
     
         ______________________________________Taxonomy of Lactobacillus plantarum DSM 9208(Morphological and physioiogical characteristics)______________________________________Characteristics of colony              Diameter of colony:(2 days, MSR Agar) &gt;1 mm, white, smoothCell form and length              Rod-shaped, different(MRS Bouillon)     lengths, single to chainsLactic acid configuration              DLGrowth at 15° C.              Reaction positiveGrowth at 45° C.              Reaction positiveEnd pH in MRS Bouillon              3.4Gas formation out of              Reaction negativeglucoseAmmonia out of arginine              Reaction negativeDiamino pimelic acid              Reaction positjve______________________________________ 
    
     The Lactobacillus brevis strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9209. 
     The complete taxonomic description for the Lactobacillus brevis strain is as follows: 
     
         ______________________________________Taxonomy of Lactobacillus brevis DSM 9209(Morphological and physiological characteristics)______________________________________Characteristics of colony:              Diameter of colony:(2 days, MSR Agar) &gt;1 mm, grey, smoothCell form and length              Rod-shaped, different(MRS Bouillon)     lengths, single to chainsLactic acid configuration              DLGrowth at 15° C.              Reaction positiveGrowth at 45° C.              Reaction slightly              positiveEnd pH in MRS Bouilion              4.4Gas formation out of              Reaction positiveglucoseAmmonia out of arginine              Reaction positiveDiamino pimelic acid              Reaction negative______________________________________ 
    
     The Pediococcus pentosaceus strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9210. 
     The complete taxonomic description for the Pediococcus pentosaceus strain is as follows: 
     
         ______________________________________Taxonomy of Pediococcus pentosaceus DSM 9210(Morphoiogical and physiological characteristics)______________________________________Characteristics of colony               Diameter of colony:(2 days, MSR Agar)  &gt;1 mm, white, smoothCell form and length               Cocci, in tetrades, also(MRS Bouilion)      at pairsLactic acid configuration               DLGrowth at 15° C.               Reaction positiveGrowth at 45° C.               Reaction positiveEnd pH in MRS Bouillon,               3.7Gas forination out of               Reaction negativeglucoseAmmonia out of arginine               Reaction positiveDiamino pimelic acid               Reaction negative______________________________________ 
    
     The Saccharomyces cerevisiae steineri strain has been deposited under the terms of the Budapest Treaty at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN Gmbh depository located at Mascheroder Weg 1b, D-3812 Braunschweig on May 19, 1994 and has been given the accession number DSM 9211. 
     The complete taxonomic description for the Saccharomyces cerevisiae steineri strain is as follows: 
     
         ______________________________________Taxonomy of Saccharomyces cerevisiae steineri strainDSM 9211(Morphological and physiological characteristics)______________________________________Yeast Morphology Agar              Form of colony:              Smooth, glossy, whole-              edge, whiteMalt Bouillon      Cell form:              Oval-shaped, multipolarly              sproutingCornmeal Agar      No pseudonycelliumMalt Agar, V8 Agar Asci formation:              2 to 4 ascospores per              ascus; ascospores being              round and smooth, ascus              formation directly out              from the vegetative cell______________________________________Assimilation and Fermentation(Percentage of positively reacting isolates)______________________________________Substrates     Fermentation                    Assimilation______________________________________Glucose        100       100Galactose      100       100Saccharose     100       100Maltose        100       100Lactose        0         0Raffinose      0         100Starch         0         0Melibiose      0         0Nitrate                  0Trehalose      0         0Cellobiose     0         0L-Arabinose    0         0D-Xylose       0         0______________________________________