Plant-based freeze protection composition

Abstract

The present invention relates to a freeze protection composition which is particularly suitable for freezing sensitive microorganisms, preferably lactobacilli or lactobacilli-containing co-cultures, to store same in the frozen state and nevertheless ensure that within 2-8 h after thawing a fast and complete reactivation of the metabolic activities takes place. The invention also relates to one-stage or multi-stage frozen storage for sourdough microbiota.

Description

[0001] Plant-based antifreeze composition

[0002] Sourdoughs, sourdough cultures and fermentatively active bacteria such as lactic acid bacteria (LAB for short) are valued worldwide as baked goods due to their unique taste and aroma and their digestibility and digestive support.

[0003] While a layperson might happily cultivate small quantities of starter cultures at home, keeping the mother culture alive in the refrigerator, multiplying it, and then producing the desired amount of bread for a typical household, the professional is required to provide starter cultures for sourdough products of impeccable quality and in any quantity. Due to the required quantities, sourdough is produced in continuous processes and on an industrial scale. Typically, liquid sourdough is produced.

[0004] Sourdoughs are demanding in their production and use, requiring a high level of expertise. To achieve ideal leavening power, sourdough cultures must be maintained in the right environment and free from contamination, for example, in order to multiply and maintain the conglomerate of various microorganisms specific to each sourdough.

[0005] In bakeries and large industrial baking facilities, this requires not only additional space and the highest standards of purity, but also, and especially, specially trained or qualified employees. For these reasons alone, sourdough is often purchased.

[0006] In its liquid state, sourdough is ready to use and is the easiest and most precise to handle. However, the shelf life of liquid sourdough is limited to approximately 5-10 days, so large operations require regular deliveries. One way to make this easier is to provide dried sourdough, which can easily be stored for a year.

[0007] An ideal situation for the baker's trade is to store liquid sourdough starters in a frozen state if they are not to be used immediately. However, it has not yet been possible to freeze and thaw lactobacilli, which are a main component of all sourdough starters, to a satisfactory extent.

[0008] Numerous publications have already addressed this problem. Lattanzi et al., 2014 (LWT- Food Science and Technology 99, pp. 948-955) summarizes that all previous attempts have been unsuccessful in using lactobacilli or lactobacilli-containing conglomerates (co-cultures), as well as, for example,

[0009] Freezing sourdough only led to the unsatisfactory result that after a freezing phase of 60 days or at the latest after 90 days the cultures were unusable and could no longer be used as a raising agent.

[0010] Thus, with previous techniques, frozen sourdough cultures cannot be fully reactivated when thawed, and freezing damage to the organisms is often observed, which leads to a loss of leavening power and the dough no longer rising or swelling.

[0011] The loss of leavening power is particularly problematic, as a sourdough culture should allow bread production without the addition of baker's yeast. However, after freezing, this is no longer the case, and baker's yeast must be added to the bread dough to achieve comparable results. W02009130219A1, for example, describes how frozen doughs often require twice the amount of yeast compared to regular doughs to achieve comparable leavening power.

[0012] It is sometimes described that a sourdough culture must be reactivated for 10-15 days after freezing until all co-cultures have recovered or re-enriched and satisfactory leavening performance can be expected. Supposedly, a sourdough stored frozen at -18°C for up to twelve months should undergo a reactivation period of 10 to 15 days before its intended use. This reactivation requires the sourdough cultures to acclimatize. For this purpose, the culture is kept from -18°C to +4°C for 24 hours, then at room temperature for a further 24 hours, and then cultivated in a water bath at 38°C with a nutrient solution, e.g., sugar or fructose. It is easy to see that industrial production does not require such reactivation phases.

[0013] Finally, there are also known methods for freezing lactobacilli or sourdough starters with the addition of glycerin. Glycerin is a trihydric alcohol that acts as an antifreeze and inhibits the formation of water crystals. However, it must be kept in mind that glycerin, as a trihydric alcohol, is an additive that not only has negative effects on the metabolism of the frozen cultures, but is also increasingly being avoided in food production.

[0014] Even freezing with glycerin does not solve all problems, and the sensory quality of baked goods made from frozen sourdough suffers during extended freezing storage. This can be recognized primarily by sensory defects such as a reduced sourdough aroma, but also by visual defects such as an overly compact, greasy breadcrumb or a crust that does not appear crispy. There are indications that the number of gas cells in the measured loaf of bread is reduced, thus reducing the elasticity of the finished baked goods.

[0015] Such influences, which arise from the frozen storage of sourdough cultures, limit the straightforward and simple processing into high-quality baked goods. Therefore, new methods and solutions are constantly being sought that enable the immediate provision or delivery of sufficiently large quantities of sourdough, as required by today's market, and that can improve, resolve, or completely avoid the problems mentioned above. Long-term storage of sourdoughs for periods of more than three months up to a full year would be desirable.

[0016] It is therefore an object of the present invention to find alternative approaches that allow lactobacilli or microbial cultures with lactobacilli, in particular e.g. sourdough, to be stored or frozen over long periods of time without impairing the aromas of the sourdough or its leavening power.

[0017] To solve this problem, the present invention provides a GSZ (glucose sulfate) that is added to the microbial coculture and / or the sourdough culture. The inventors were able to demonstrate that sourdoughs or individual or sensitive LAB strains that were mixed with the GSZ according to the invention and then frozen retained their essential properties over periods of 30, 60, 90, and up to 120 days of frozen storage, and, in particular, demonstrated full leavening power, full metabolic activity, and / or full gas evolution after a reactivation time of only 4-8 hours.

[0018] The antifreeze composition according to the invention (hereinafter abbreviated as GSZ) for single freezing and / or multiple refreezing for microbial cocultures containing lactobacilli and / or sourdough or sourdough starter contains

[0019] • 5 - 65 wt% biopolymers and / or biological hydrocolloids;

[0020] • 7 - 55 wt.% plant and / or microbial mucilage

[0021] • 10 - 45 wt% proteins;

[0022] • 1 - 10% by weight sugar;

[0023] • and

[0024] • 0.1 - 5 wt% free amino acids.

[0025] The said GSZ is particularly suitable for improving the freezing and thawing process, and thus the frozen storage of microbial cultures containing lactobacilli, such as sourdough or LAB pure cultures, which are often considered to be very sensitive.

[0026] The term "microbial cultures" in the context of this application is to be understood as a conglomerate or microbiota of several bacterial strains, all of which live together and alongside one another in this coculture and do not displace or overgrow each other. Sourdough in general is an example of this, and thus the term "sourdough" in the context of this application is to be understood as a conglomerate or microbiota of numerous bacterial strains, including a wide variety of organisms, for example, from the Lactobacillus family, and a wide variety of organisms commonly referred to as yeasts, all of which—if properly managed—live, survive, and reproduce in this biomass.

[0027] The term "sourdough" also describes a dough that has been created or produced with the aid of sourdough cultures, also called sourdough starter, or a conglomerate of microorganisms as described above. Such a dough has a high acid content, since LABs are capable of producing, among other things, lactic and acetic acid. In the context of this application, the term "sourdough starter" is also to be understood as a smaller amount of the same sourdough or sourdough conglomerate, which is then used directly to produce another dough or is added to a flour mixture for this purpose.

[0028] In the context of this invention, the freezing and thawing process, and thus the frozen storage of such microbial cocultures containing lactobacilli and / or sourdough or sourdough starter, is particularly improved. To demonstrate such an improvement, corresponding cocultures and combinations of pure strains were first mixed with the composition according to the invention, but also with numerous other compositions for comparison purposes, and frozen at least once, preferably several times. The frozen storage was carried out for various periods of time, and subsequently, the metabolic activity (vitality values) of the thawed cultures was analyzed. For this purpose, a viable microbial count was determined, among other things.However, in the context of the invention, the number of viable bacteria is less important, because the essential properties that determine whether thawed sourdough cultures are suitable for use in the baking process are measured by metabolically measurable parameters.

[0029] In the context of this application, the metabolic parameters to be recorded are therefore of greater importance, since they indicate that sourdough cultures and / or pure or co-cultures containing lactobacilli are alive and metabolically active and, for example, show increasing gas evolution and increasing acid formation, since lactobacilli are capable of producing lactic and acetic acid, for example.

[0030] The inventors were able to show that, when using the GSZ according to the invention, an outstanding improvement and extension of frozen storage of sourdough and / or microbial cocultures containing lactobacilli could be achieved, particularly with regard to acid development, gas volume development and thus leavening power in the dough.

[0031] The antifreeze composition used for this purpose is a composition that contains various components in coordinated weight ratios - as specified above - whereby the essential component, namely the plant and / or microbial mucilages of the antifreeze composition, are water-soluble.

[0032] In the context of this application, "plant and / or microbial mucilages" mean a selection of purified, isolated, and / or natural substances contained in plants or microorganisms that have the property of swelling upon contact with water. These are regularly referred to as mucilages, which in the case of plant mucilages consist of, for example, heteropolysaccharides and are selected from the group of pentosan, galactan, galactan sulfate, pectin, arabinoxylan, arabinogalactan, glucan, levan, and dextran. In this invention, plant mucilages are preferably used, which consist of, for example, galactan sulfates and polyuronides such as pectins and often form colloids or hydrocolloids or even gels or hydrogels, which derive their name from their mucilaginous, highly viscous, and elastic consistency.

[0033] Since plant mucilages are also contained in cereal grains and seeds, the present invention uses not only purified mucilages, but also flours from ground seeds, namely, for example, flax, linseed, quinoa, chia, pulses (galactomannans), tamarinds, fenugreek, psyllium, carob seeds, pomegranate seeds, ground oilseeds such as pumpkin, sunflower, hemp or rapeseed, and coffee beans.

[0034] According to the present invention, the preferred and interchangeable plant and / or microbial mucilages are also selected from the group comprising pentosan, galactan, galactan sulfate, pectin, arabinoxylan, arabinogalactan, glucan, levan, dextran and mixtures thereof.

[0035] The plant and / or microbial mucilages ensure controlled water retention in the GSZ, thus improving, in particular, a slower freezing process and a subsequent, slower water-releasing thawing process. This delay has a very positive effect on the metabolic activity of the frozen and thawed microorganisms. It has been shown that a particularly high content of these mucilages, namely more than 7, 10, 15, 17, 20, or 25 wt.% and up to 30, 35, 40, 45, 50, or 55 wt.% of the antifreeze composition, improves the freezing capacity of the microbial cocultures and, in particular, allows for rapid reactivation of metabolic activity, typically after just 2, 4, 6, or 8 hours after thawing.

[0036] Furthermore, the antifreeze composition contains purified, isolated and / or plant-derived proteins, which are preferably selected from the group comprising purified wheat protein, gluten, purified legume protein, pea protein and mixtures thereof.

[0037] The plant proteins lead to increased concentration during freezing due to the removal of water, and the antifreeze composition develops a buffering function through the proteins, balancing any pH fluctuations that occur, thus preventing pH-related agglomerations. In the inventive composition, the plant proteins in particular have a positive effect on agglomeration behavior. Nevertheless, the plant proteins can also be replaced with skimmed milk powder and casein peptone.

[0038] It has been shown that in particular a content of 10 to 45 wt.% of protein in the antifreeze composition, namely 10 wt.% and more than 10 wt.%, namely 11, 12, 13, 14, 15, 17, 20, 22, 25 or 28 wt.%, as well as up to 30, 35, 40 or 45 wt.% protein in the antifreeze composition, significantly stabilizes the freezing and single or multiple thawing process.

[0039] A further component of the antifreeze composition are purified, isolated and / or plant-occurring sugars, which are added to or contained in the antifreeze composition in small amounts and which are selected from the group of water-soluble monosaccharides and polysaccharides, namely galactose, maltose, oligofructose, dextrose, arabinose, tetrahydrocannabinol and mixtures of the above.

[0040] The selected sugars, and especially T-rehalose, alter the osmotic pressure in and around the microorganisms during freezing. This slowed exchange can reduce crystal formation during freezing, thus preserving the MOs and demonstrating better viability after thawing.

[0041] It has been shown that in particular a content of 1 to 10 wt.% of sugar in the antifreeze composition, namely 1, 2, 3, 4, 5, wt.% and further 6, 7, 8, 9 and up to 10 wt.% sugar in the antifreeze composition, significantly improves the survivability of the MO during the freezing and single or multiple thawing process.

[0042] The antifreeze composition also contains biopolymers. The term "biopolymer" in the context of the application refers to a carbohydrate that primarily exists as a starch or hydrocolloid. In particular, the application refers to starch from rice, corn, potatoes, or wheat, but also from tapioca, for example, as a biopolymer.

[0043] The optional addition of biopolymer to the composition according to the invention improves handling and processability, but does not directly improve the freezing and thawing process. It has been shown that a proportion of 5 to 65 wt.% of biopolymer and / or isolated or purified starch in the GC is suitable for facilitating the creation of a crumbly consistency of the microorganisms to be frozen together with the GC. Depending on the residual water content of the microorganism cultures to be frozen, it is advisable to adjust the proportion of biopolymer and to add either no biopolymer or 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or even 65 wt.% biopolymer to the GSZ, which are selected from the group of biological hydrocolloids, for example ground seeds of fenugreek, linseed, chia seeds, psyllium seeds, carob tree and mixtures of the aforementioned.

[0044] Interestingly, the specified percentage composition can be achieved by selecting suitable flours. The examples show that, for example, a GSZ consisting of 40% chia flour or linseed flour and 60% rice flour falls within the claimed range (see Example 1, Recipes V3, V4, V5), and this can be modified within the limits of the claim by changing the mixing ratio (see Examples 9, 10, and 11).

[0045] Particularly interesting is a GSZ consisting solely of flaxseed flour, which corresponds to a biopolymer / protein ratio of approximately 40 / 40, thus representing a simple but effective composition. See especially V11A.

[0046] Finally, it has been shown that the antifreeze composition can also contain small amounts of free amino acids. It is assumed that these have a protective effect on the metabolically active proteins of the microbial cocultures and thus contribute to the rapid reactivation of metabolic functions. If free amino acids are added at all, this occurs only in small amounts, preferably only 0.5 wt.%, optionally 1 or 1.5 wt.%, alternatively up to 2, 3, 4, and a maximum of 5 wt.%, with the free amino acids being selected from the group consisting of proline, glutamates, arginine, alanine, and mixtures thereof. During drying, the amino acids support the equilibrium between internal (cell) pressure and external (cell) pressure, so that the free amino acids are regarded as molecular spacers.

[0047] While the present invention particularly proposes a GSZ (freeze protection composition) blended from the above-mentioned components, thus ensuring clear reproducibility of the recipe, it has been shown that antifreeze compositions consisting of flour-like compositions or premixed flour-like compositions are also suitable for correspondingly improving frozen storage. Typically, flours or flour-like compositions are selected for this purpose that also have a correspondingly high proportion of plant mucilage and that are selected, for example, from the group of flours containing rice flour, linseed flour, quinoa flour, buckwheat flour, corn flour, chia flour, chickpea flour, lentil flour, pea flour, pomegranate seed flour, coffee bean flour, oilseed presscakes ground to flour, namely pumpkin, sunflower, hemp, or rapeseed, as well as mixtures of the aforementioned.

[0048] In terms of processability, rice flour starch, corn starch, potato starch, and even tapioca have a positive effect on the antifreeze composition, as does, for example, rice flour. If the flour-like composition does not contain a desired amount of mucilage, namely a proportion of 7, 10, 15, 20, 25, 30, 35, 40, 45, or 55 wt.%, it can be enriched according to the desired ratio. For this purpose, either isolated plant mucilage or purified microbial mucilage can be added.

[0049] According to the examples given below, linseed flour / linseed flour and / or chia flour are particularly suitable as starting materials for the antifreeze composition according to the invention due to their high intrinsic mucilage content.

[0050] According to further embodiments, particularly when using linseed flour and / or chia flour, a composition of the ingredients within the scope of the GSZ according to the invention can be detected.

[0051] For example, a GSZ based on flaxseed flour has the following composition, namely 30-45% flaxseed flour (or chia flour) and 55-70% rice flour, thus achieving a percentage composition of:

[0052] • 7-55 wt.% vegetable mucilage,

[0053] • 1-5% w / w sugar,

[0054] • 10-45% w / w proteins,

[0055] • 0.1-4 wt% free amino acids.

[0056] For improved processability, rice flour or rice starch is added to the linseed flour in a ratio of 1:1 to 1:3, according to other embodiments. The added rice flour brings the percentage of the total composition to 100%.

[0057] What is crucial when adding rice flour is not just the addition of the percentages, but also the ability of rice flour to bind water quickly and effectively and thus, when mixed with or sprinkled into the microbial culture, to create the preferred crumbly consistency of the microbial culture to be frozen.

[0058] For the purpose of freeze-preserving a microbial coculture of lactobacilli and yeast or sourdough, the GSZ according to the previously described embodiments is first mixed with this sourdough or the corresponding coculture, typically in a microbial GSZ ratio of 1:1 to 1:5.

[0059] According to the present application, the sourdough or microbial culture is first cooled to a temperature between 18°C ​​and 5°C and then mixed with 10-50 wt.% of the antifreeze composition described above. The amount of GSZ added depends crucially on the water content of the microbial coculture or sourdough or sourdough starter. In any case, sufficient GSZ should be added so that the mixture of microbial coculture and GSZ has a crumbly consistency.

[0060] In the context of this application, "crumbly consistency" refers to semi-moist granules whose individual granules can be broken down by applying minimal mechanical force. The term "crumbly consistency" therefore describes the consistency of the mixture of microorganisms and a flour-like composition that is no longer wet, moist, or sticky, but rather breaks down into small dough conglomerates, which are, for example, dusted with flour on the outside, and therefore have a crumbly, loose consistency. This step also distinguishes the process according to the invention from known processes for frozen storage, which take place either in the lyophilized or liquid state.

[0061] This mixture with a crumbly consistency can then be further cooled, or even deep frozen, typically at -12° to -20°C, or flash frozen at typically -30°C.

[0062] This mixture allows loss-free frozen storage of sourdough or sourdough starter for well over 30, 60 or 90 days and ultimately up to 180 days, thus enabling maximum flexibility in industrial baking operations.

[0063] It is particularly noteworthy that the freezing composition according to the invention, in addition to its significantly improved storage life, also has the advantage of not containing any added glycerin. Glycerin has long been used to freeze microbial cultures and to achieve cellular cryoprotection by reducing water crystal formation. Glycerin is an additive and is listed under the number E422.

[0064] For natural foods, it would be desirable to be able to do without such additives. This invention makes it possible for the first time to do without glycerin as an additive without having to accept a reduction in shelf life.

[0065] If the addition of glycerin does not raise concerns, the frozen storage achieved by the GSZ of the present invention can be further improved by adding more glycerin. For this purpose, 0 to 2 wt.%, 1 to 3 wt.%, 2 to 4 wt.%, 3 to 5 wt.%, 5 to 7 wt.%, 4 to 8 wt.%, or 7 to 10 wt.% of glycerin can be added to the GSZ according to the invention without significantly impairing the leavening performance or the measurable metabolic activity of the thawed sourdoughs.

[0066] The cryopreservation method described herein is particularly suitable for the frozen storage of microbial cocultures containing highly sensitive microorganisms. These include lactobacilli selected from the group consisting of lactobacilli, companilactobacilli, fructilactobacilli, furfurilactobacilli, lacticasei bacteria, lactipla ntibacilli, lentilactobacilli, levilactobacilli, limosilactobacilli, lactococci, leuconostocci, pediococci, Weissella, and yeasts selected from the group consisting of Kazakhstania, Candida, Saccharomyces, Dekkera, Kluyveromyces, Torulaspora, and Wickerhamomyces.

[0067] Sourdough, sourdough microbiota, or sourdough starter also contain one or more of the lactobacilli and yeasts listed here; therefore, the method described herein and the GSZ according to the invention are particularly suitable for improving the frozen storage of sourdough or sourdough starter.

[0068] Accordingly, the inventors were able to demonstrate that sourdough frozen and stored in the freezer according to the method presented here and with the inventive GSC exhibits excellent metabolic reactivation within 1-10 hours after thawing. For example, gas fermentation with a gas volume development of at least 200 ml up to 600 ml was achieved just a few hours after thawing, which would be sufficient to represent the full leavening power expected from a sourdough. It was also shown that sourdough frozen and stored in the freezer according to the method presented here and with the inventive GSC achieves acidification of a starter to pH values ​​of at least 5.5 to 3.5 within 1-10 hours after thawing. This is clear evidence of the metabolic activity or reactivation of the thawed lactobacilli.

[0069] The process described herein is particularly advantageous for the production of frozen sourdough, which can then be stored, transported, or sold for long periods even in the frozen state. Said frozen sourdough is characterized by the fact that the microbial coculture contains, in particular, sensitive microorganisms selected from the group consisting of lactobacilli, companilactobacilli, fructilactobacilli, furfurilactobacilli, lacticaseibacilli, lactiplantibacilli, lentilactobacilli, levilactobacilli, limosilactobacilli, lactococci, leuconostocci, pediococci, Weissella, and mixtures thereof, and which are rapidly reactive after thawing and remain metabolically active.

[0070] According to further embodiments, the frozen sourdough treated with the freeze protection according to the invention contains in particular lactobacilli, which are selected from the group containing L. acidophilus, L. amylovorus, L. crispatus, L. helveticus, L. johnsonii, Co. alimentarius, Co. crustorum, Co. mindensis, Co. paraalimentarius, Fr. fructivorans, Fr. lindneri, Fr. sanfranciscensis, Fu. rossiae, Fu. siliginis, Lc. paracasei, Lp. plantarum, Lp. pentosus, Le. buchneri, Le. parabuchneri, Lv. brevis, Lv. cerevisiae, Lv. hammesii, Li. fermentum, Li. frumenti, Li. mucosae, Li. panis, Li. pontis, Li. reuteri, Li. secaliphilus, L. lactis, L. citreum, P. acidilactici, P. parvulus, P. pentosaceus, M / . ciborio, M / . confusa, M / .paramesenteroides and mixtures thereof and further contains yeasts selected from the group consisting of Kazachstania humilis, Candida milled, Candida humilis, Kazachstania exigua, Saccharomyces exigua, Dekkera bruxellensis, Kazachstania unispora, Saccharomyces unisporus, Kluyveromyces lactis, Saccharomyces boyonus, Saccharomyces cerevisiae, Saccharomyces postorionus, Torulaspora delbrueckii, Torulosporo pretoriensis, Wickerhomomyces anomalus and mixtures thereof.

[0071] As shown in the following examples, viable bacterial counts were initially determined for the samples subjected to freezing tests. The data clearly show that a significant improvement in survival rates was initially demonstrated for lactobacilli (LABs), which are sensitive to freezing tests.

[0072] For example, Example 5, Table 3, shows that in experiments that included a first freezing, then a simulated "transport" (i.e., a 5-day freeze-out period, followed by a second freezing), the number of surviving LABs and yeast decreased, but still remained at a level that allowed for rapid regeneration. This trend persisted even when the frozen storage period was extended.

[0073] Furthermore, it is shown that the proposed freezing method offers significant improvements compared to previous long-term storage trials, particularly in the frozen storage of selected LAB cultures and sourdough strains. Furthermore, the following examples demonstrate that when checking the activity parameters after thawing, not only was the survival of the sensitive cultures improved, but in particular, the regeneration or reactivation time until full metabolic activity was detected was significantly improved.

[0074] In corresponding reactivation tests after frozen storage, the acid development in thawed reactivated cultures and sourdough starters was measured within the scope of the present invention, as acid formation is an important parameter of lactobacillus metabolism; on the other hand, the resulting gas volume and thus the leavening power were also measured as a function of the fermentation time.

[0075] In particular, the inventors were able to demonstrate that sourdoughs frozen according to the present invention not only contain viable LAB, but also show a rapid reactivation of the fermentation performance and thus meet the expectations of the industrial bakers and baking industry, namely to achieve reactivation and thus readiness for use in a few hours.

[0076] Character description

[0077] Figure 1 shows the pH value curves of the above-mentioned recipes VI sourdough starter without GSC (solid line) and V3 sourdough starter with plant-based GSC (dashed line) in the observed time course from 0 to 20 hours.

[0078] Figure 2 shows the pH value curves of the above-mentioned recipes VI sourdough starter without GSZ (solid line), V3 sourdough starter with GSZ (dashed line), V4 sourdough starter with GSZ based on linseed flour and glycerol addition (short lines), V5 sourdough starter with GSZ based on chia flour (short line 2x dots) and V6 sourdough starter with GSZ based on chia flour and glycerol addition (long line 2x short line) in the observed time course from 0 to 20 hours.

[0079] Figure 3 shows the pH values ​​of the reference sample of a non-frozen starter consortium (microbiota A), which in the context of the application is regularly identified as a sourdough starter “fresh” or “frozen” and is presented in a corresponding sample with frozen storage at different storage times in order to record the acid development under suitable fermentation conditions over a period of up to 20 hours.

[0080] Figure 4 shows the measurement results of a gas volume measuring device that records the CO2 produced in mL / g at 28°C over a period of up to 20 hours during the fermentation process in the homogeneous sourdoughs VI and V3.

[0081] Figure 5 shows the measurement results of a gas volume measuring device which records the CO2 produced in mL / g at 28°C over a period of up to 20 hours for the fermentation process in the homogeneous sourdoughs VI, V3, V4, V5 and V6.

[0082] Figure 6 shows the results of the determination of the fermentation height in the form of the volume increase in mL of the homogeneous sourdough samples VI and V3, which were filled into a cylinder and measured at 32°C and a relative humidity of 75% for a period of up to 750 minutes. Figure 7 shows the results of the determination of the fermentation height in the form of the volume increase in mL of the homogeneous sourdough samples VI, V3, V4, V5, and V6, each of which was filled into a cylinder and measured at 32°C and a relative humidity of 75% for a period of up to 750 minutes.

[0083] Figure 8 A and B show the results of acid development and gas quantity development within a few hours for the microbiota B treated with the GSZ according to the invention and thawed after 3 months.

[0084] Examples:

[0085] The freezing experiments described below illustrate the influence of the antifreeze compositions on selected cocultures, e.g. so-called sourdough starter cultures, as well as their influence on the stability of the viable bacteria counts, the reactivation of metabolic activities and the fermentation performance of frozen and subsequently thawed sourdough starter cultures.

[0086] In the following experiments, different sourdough starter cultures from different cocultures and with different sourdough microbiota are used:

[0087] Microbiota A consists of at least, but not exclusively, the following Saccharomyces pastorianus, Lacticaseibacillus paracasei, Limosiloctobocillus fermentum Loctiplontibocillus plontorum Lactobacillus helveticus, Companilactobacillus paralimentaarius, Leuconostoc lactis.

[0088] Microbiota B consists of at least, but not exclusively, the following Kazachstania unisporo and Fructolactobacillus sonfronciscensis.

[0089] Microbiota C consists of at least, but not exclusively, the following Saccharomyces cerevisioe, Limosiloctobocillus pontis and Lactobacillus helveticus.

[0090] Microbiota D consists of at least, but not exclusively, the following Kazachstania humilis and Fructilactobacillus sonfronciscensis.

[0091] Example 1

[0092] Freezing test with sourdough starter culture

[0093] To test the effects of frozen storage on selected sourdough starter cultures, the sourdough starter culture was observed once with the addition of a plant-based GSZ according to Example 1 and once without the addition of a comparable antifreeze mixture.

[0094] LA - Conventional freezing test

[0095] For the antifreeze-free control, the selected sourdough starter cultures were first mixed with recipe VI, fermented for 5-12 hours at 4°C, then packaged in easily processable 200 g portion sizes in plastic containers as test doughs, and after a further maturation of 1.5-10 hours at 28°C, the containers were immediately subjected to shock freezing at -30°C.

[0096] They were then frozen for the respective test periods of 10, 30, 60, 90 and 120 days and subjected to two freezing and two thawing processes each.

[0097] For this experiment, which represents the current practice of freezing test doughs, the test doughs were mixed with a composition according to Recipe VI (composition described below): lB - Freezing test with GSZ with linseed flour In parallel, a comparable test procedure was chosen, but this time with the addition of the inventive plant-based GSZ according to recipe V3, which differs in that after the first fermentation at 4°C, the cooled sourdough mixture was mixed with the GSZ, resulting in a crumbly consistency.

[0098] The crumbly mixture (1:1) of sourdough and GSZ was allowed to mature for 1.5 hours at 28°C, mixed again, and packaged in 200g plastic containers, which were then matured for 8.5-10 hours at 28°C and then shock-frozen at -30°C. LC - Freezing test with GSZ with linseed flour and additional glycerin.

[0099] After fermentation, the sourdough was cooled to 4°C and then mixed with the plant-based gluten-free flour (GSZ) and glycerin to a crumbly consistency. The mixture was then allowed to mature for 1.5 hours at 28°C, mixed again, and packaged in 200-g plastic containers, which were then matured for 8.5–10 hours at 28°C and then shock-frozen at -30°C. ID Freezing test with gluten-free flour (GSZ) and chia flour

[0100] After fermentation, the sourdough was cooled to 4°C and then mixed with the plant-based chia flour-based GSZ to a crumbly consistency. The mixture was then allowed to mature for 1.5 hours at 28°C, mixed again, and packaged in 200g plastic containers. These were then matured for 8.5-10 hours at 28°C and then flash frozen at -30°C.

[0101] LE - Freezing test with GSZ with chia flour and glycerin addition

[0102] Recipe V6

[0103] After fermentation, the sourdough was cooled to 4°C and then mixed with the plant-based chia flour-based GSZ to a crumbly consistency. The mixture was then allowed to mature for 1.5 hours at 28°C, mixed again, and packaged in 200g plastic containers. These were then matured for 8.5-10 hours at 28°C and then flash frozen at -30°C.

[0104] Example 2:

[0105] Metabolic activities of thawed cocultures - acid formation

[0106] 2.A: Experimental description for acid development with VI and V3

[0107] Using the sourdough starter cultures frozen with VI and V3, the acid development was recorded to demonstrate the reactivation and viability of the thawed microorganisms in the sourdough.

[0108] For this purpose, homogeneous sourdoughs were prepared from 30 g VI or V3, 150 g wheat flour type 1050 and 112.5 g water.

[0109] In these sourdoughs, the acid development was recorded in the form of a pH curve at 28°C and over a period of 20 hours.

[0110] A recording of the acid development using a pH logger is shown in Figure 1 for the homogeneous sourdoughs VI and V3 at 28°C. This shows that in experiment VI without the addition of the GSZ, the curve initially rises at 0 hours, starting at a pH of 5.3, slowly becoming less acidic, until a maximum pH of 5.5 is reached after about 5 hours and remains constant for about 12.5 hours. After that, there is a sigmoidal decrease in the pH up to 20 hours, at which point a significantly more acidic pH of 3.8 is reached. Therefore, viewed over the period, an increase in acid development, and thus a sign of active sourdough work / fermentation performance, is only visible from about 11-12.5 hours.

[0111] In experiment V3, the sourdough starter culture frozen with plant-based GSZ started at a pH range of approximately 5.45 and the pH then fell sigmoidally after about 2.5 hours and reached a more acidic pH of 4.5 after about 7 hours. The value continued to fall steadily until, at the end of 20 hours, the pH fell to the most acidic pH of 3.8.

[0112] This is a clear indication of the strong metabolic activity of the sourdough starter culture, which can be achieved despite freezing. Thus, sourdough cultures supplemented with the GSZ according to the invention are usable after a relatively short activation / regeneration time and reach almost full fermentation performance very quickly.

[0113] In a direct comparison of VI to V3, it becomes clear that the GSZ leads to a significant improvement in the regeneration after the thawing phase and thus to a completely surprisingly more active metabolic behavior than would be the case with a sourdough culture without the stabilizing and positive influence of the antifreeze compositions on the fermentation performance.

[0114] Adding substances containing the GSZ according to the invention to the sourdough culture before freezing and deep-freezing storage leads to such significantly improved results that long-term storage is now also conceivable, thus significantly improving the flexibility of use for the baking industry.

[0115] 2.B: Experiment description for acid development with VI, V3, V4, V5, V6

[0116] Likewise, acid development was recorded using sourdough starter cultures frozen with VI, V3, V4, V5 and V6 to demonstrate the reactivation and viability of the thawed microorganisms in the sourdough.

[0117] For this purpose, homogeneous sourdoughs were prepared from 30 g of VI or V3, or V4, or V5 or V6, 150 g of wheat flour type 1050 and 112.5 g of water.

[0118] In these sourdoughs, the acid development was recorded in the form of a pH curve at 28°C and over a period of 20 hours.

[0119] The pH values ​​of the homogeneous sourdoughs VI, V3, V4, V5 and V6 were recorded to determine the acid development over a period of up to 20 hours (see Fig. 2).

[0120] For VI, the curve initially rises slowly from 0 hours at a pH of 5.3, becoming less acidic until a pH of 5.5 is reached after about 5 hours, and this remains constant for about 12.5 hours. After that, there is a sigmoidal decline in the pH until 20 hours, when a significantly more acidic pH of 3.8 is reached. Thus, viewed over the period, an increase in acid development, a sign of active sourdough work / fermentation, is only visible from about 11-12.5 hours.

[0121] For V3, the measured acidity starts at a pH of approximately 5.45, which then begins a sigmoidal decline after about 2.5 hours, reaching a more acidic pH of 4.5 after about 7 hours, and continues to decline steadily until, finally, after 20 hours, the pH drops to 3.8. This is a clear indication of the strong metabolic activity of the thawed sourdough starter culture, even after a very short activation / regeneration time, leading to full fermentation performance.

[0122] Recipe V4: Sourdough starter with GSZ based on linseed flour and glycerin addition starts at 0 hours of the curve with a pH of 5.5, increases to approximately 5.7 after the start of the test, and then decreases sigmoidally. After 10 hours, acid development reaches a pH of 4.2 and after 20 hours, a pH of 3.8. The addition of linseed flour and glycerin shows that the combination of GSZ and glycerin also has a positive effect on the acid development of the sourdough culture and leads to faster reactivation of the sourdoughs than is the case with sourdough recipes without antifreeze compounds.

[0123] In V5 sourdough starter with chia flour-based GSZ, the pH starts at 5.5 at 0 hours, increases to a pH of 5.7 after approximately 2.5 hours, and then decreases sigmoidally over the entire period, reaching a pH of approximately 3.75 after 20 hours. Thus, V5 reaches a more acidic pH more quickly than the other sourdough cultures tested.

[0124] Recipe V6, a sourdough starter with chia flour-based GSZ and added glycerin, clearly shows that at 0 hours, the pH measurement starts at a pH of 5.5. After approximately 2.5 hours, the pH drops below this value, and acid production begins. In the sigmoidal curve, the pH decreases over the entire 20-hour period. After 10 hours, a pH of 4.0 is reached.

[0125] Therefore, the use of a GSZ based on chia flour (without glycerin) is just as effective as the comparable GSZ based on chia flour with the addition of glycerin.

[0126] Example 3:

[0127] Metabolic activities of thawed cocultures - gas formation

[0128] 3.A - Experiment description for gas volume development with VI and V3

[0129] Using the sourdough starter cultures frozen with VI and V3, the gas volume released by the MO was measured to demonstrate the reactivation and viability of the thawed microorganisms (MO) in the sourdough. The amount of carbon dioxide formed was measured using the methods described in utility model DE202010005307U1. For this purpose, homogeneous sourdoughs were prepared from 30 g of VI or V3, 150 g of type 1050 wheat flour, and 112.5 g of water.

[0130] The formation of CO2 as a product of metabolic fermentation was recorded in mL / g using a gas volume meter over a period of 20 hours.

[0131] To record the gas volume development of VI and V3 during fermentation, the CO2 produced in mL / g at 28°C was recorded over a period of up to 20 hours using a gas volume meter in the homogeneous sourdoughs VI and V3 (see Fig.: 4).

[0132] Figure 4 shows the gas volume generated by formulations VI (solid line) and V3 (dashed line) over a time course of 20 hours, with VI starting at 0 hours with 0.0 mL / g CO2 gas evolution and showing a slightly increasing gas evolution up to the final volume of 1.25 mL / g CO2 at the end of 20 hours.

[0133] V3 (dashed line) also starts at 0 hours at 0.0 mL / g CO2 gas evolution, then increases slightly to a gas evolution of 0.4 mL / g up to about 5 hours and then increases over the next 5 hours to 1.6 mL / g and then decreases somewhat more slowly to 2.15 mL / g CO2 gas evolution up to 15 hours and continues to flatten out to the final value of 2.4 mL / g up to 20 hours.

[0134] V3 thus has a doubling of the CO2 gas volume compared to VI, thus the addition of the GSZ leads to twice the metabolic turnover and a corresponding gas evolution compared to a sourdough culture without the addition of an antifreeze composition.

[0135] 3.B - Experiment description for gas volume development with VI, V3, V4, V5, and V6

[0136] Using the sourdough starter cultures frozen with VI or V3, or V4, or V5 and V6, the gas volume released by the MO was recorded to demonstrate the reactivation and viability of the thawed microorganisms (MO) in the sourdough.

[0137] For this purpose, homogeneous sourdoughs were prepared from 30 g of VI or V3, or V4, or V5, or V6, 150 g of wheat flour type 1050 and 112.5 g of water. The formation of CO2 as a product of metabolic fermentation was recorded in mL / g using a gas volume meter over a period of 20 hours.

[0138] Furthermore, during the fermentation process in the homogeneous sourdoughs VI, V3, V4, V5 and V6, the CO2 produced was measured in mL / g at 28°C over a period of up to 20 hours using a gas volume meter (see Fig.: 5).

[0139] In Figure 5, the curves of the resulting CO2 gas volume are plotted against the test time up to the end point after 20 hours for VI (solid line) sourdough starter without freeze protection, V3 (long-dashed line) sourdough starter with vegetable freeze protection, V4 (short-dashed line) sourdough starter with vegetable GSZ based on linseed flour with glycerol addition and V5 (-..line) sourdough starter with GSZ based on chia flour and V6 sourdough starter consortium 2 with vegetable GSZ based on chia flour with glycerol ( line).

[0140] VI starts at 0 hours with 0.0 mL / g CO2 gas evolution and reaches a final volume of 1.25 mL / g CO2 by the end time of 20 hours with a slightly increasing gas evolution.

[0141] V3 (long dashed line) starts at time 0 hours at 0.0 mL / g CO2 gas evolution, increases slightly to a gas evolution of 0.4 mL / g up to about 5 hours, then increases over the next 5 hours to 1.6 mL / g, then decreases somewhat more slowly to 2.15 mL / g CO2 gas evolution up to 15 hours, and continues to flatten out to the final value of 2.4 mL / g CO2 gas evolution up to 20 hours, representing a high CO2 gas evolution.

[0142] V4 (short dashed line) starts at time 0 hours at 0.0 mL / g CO2 gas evolution, then increases very slowly over the course of 20 hours, initially up to 5 hours, to approx. 0.4 mL / g CO2 gas evolution, and then reaches CO2 gas volumes of 1.45, 2.0 and 2.3 mL / g up to 10 hours, 15 hours and 20 hours, respectively.

[0143] V5 (-..line) starts at 0 hours with 0.0 mL / g CO2 gas evolution and reaches only 0.25 mL / g CO2 gas evolution after about 7 hours. Only from this point on does the CO2 gas volume generation increase. After 10 hours, 1.1 mL / g is reached. After 15 hours, 1.8 mL / g is reached, and the final value after 20 hours is 2.2 mL / g CO2 gas evolution.

[0144] V6 (dotted line) starts at 0 hours with 0.0 mL / g CO2 gas evolution and, after a moderate increase after 5 hours, reaches a gas volume of approximately 0.55 mL / g CO2. Thus, V6 has the highest initial value of all compared curves. Even after 10 hours, the evolved gas volume remains the highest of all curves at approximately 1.9 mL / g CO2. V6 continues to increase the most in evolved gas volume of all curves until the end after 20 hours, reaching 2.85 mL / g CO2 at the end point.

[0145] The results illustrate that without the addition of a GSZ, only minimal gas evolution occurs, and consequently, only a small volume of CO2 gas is measured. This reflects the fact that even less active cultures can initiate a fermentation process if sufficiently long incubation times are maintained. Even though V5 shows a delayed increase in CO2 gas production, V5, together with V3 and V4, clearly demonstrates that suitable antifreeze compositions allow for a significant improvement in metabolic activities after frozen storage of sourdough starters, especially in direct comparison with frozen storage of sourdough cultures without antifreeze composition. V6 shows consistently high CO2 gas evolution throughout the entire measurement period.

[0146] Example 4:

[0147] Experimental description of fermentation height

[0148] To demonstrate the activity of the sourdoughs, the fermentation height was determined during fermentation. The fermentation performance of the dough pieces and sourdough starter cultures was observed at 32°C and a relative humidity of 75%.

[0149] 4.A: Description of the test for fermentation height with VI or V3

[0150] For this purpose, homogeneous sourdoughs were prepared from 30 g VI or V3, 150 g wheat flour type 1050 and 112.5 g water.

[0151] The homogeneous sourdoughs were transferred into a cylinder in such a way that they contained no additional air pockets. Furthermore, the surface was pressed flat to ensure the most compact dough structure possible at the beginning of the measurement.

[0152] To demonstrate the activity and leavening power of the sourdoughs, the fermentation height was determined during fermentation. The fermentation performance of the dough pieces and sourdough starter cultures was measured at 32°C and a relative humidity of 75% (Figure 6).

[0153] Figure 6 shows that with VI (round filled dots) after the start of the experiment at time 0 minutes with a volume of 270 mL, no further volume increase beyond this value can be achieved even after 750 minutes.

[0154] V3 (open circle), on the other hand, also starts at 270 mL at time 0 minutes and develops an almost linear increase in volume from 180 minutes up to 570 mL at the test time point at 400 minutes, reaching a volume of approximately 600 mL at 600 minutes. A stable volume of 580 mL is reached at the end point of the measurement.

[0155] While VI appears inactive, V3 can achieve a doubling to almost tripling of the fermentation height, clearly demonstrating that the appropriate GSZ allows for a significant improvement in metabolic activities after frozen storage of sourdough starters, thus not only enabling but also improving the use of frozen sourdoughs. Furthermore, homogeneous sourdoughs were prepared from 30 g of VI or V3, or V4, or V5, or V6, 150 g of type 1050 wheat flour, and 112.5 g of water.

[0156] To determine the fermentation height during fermentation, the volume in mL of a homogeneous sourdough sample VI, V3, V4, V5 and V6, which was filled into a cylinder, was measured at 32°C and a relative humidity of 75% for a period of up to 750 minutes (see Fig.: 7).

[0157] From Figure 7 it can also be seen that VI (round filled dots) after the start of the experiment at time 0 minutes with a volume of 270 mL, no further volume increase beyond this value can be achieved even after 750 minutes.

[0158] V3 (open circle), on the other hand, also starts at 270 mL at time 0 minutes and develops an almost linear increase in volume from 180 minutes until reaching 570 mL at the test time point of 400 minutes. After that, the volume value stagnates and only reaches a volume of approximately 600 mL at 600 minutes. A stable 580 mL is reached at the end point of the measurement.

[0159] V4 (filled triangle with the tip facing down) shows an initial fermentation height of 270 mL at 0 minutes. After 180 minutes, there is barely any increase in the fermentation height, only after that does a noticeable increase occur, for example, at 300 minutes, the fermentation height is 340 mL. After 400 minutes, 530 mL is reached. After 620 minutes, a fermentation height of 580 mL is reached. Towards the end of the experiment, at 750 minutes, a stable volume of approximately 560 mL is reached.

[0160] V5 (empty triangle with apex pointing upward) shows similar initial behavior to V4, but after the starting volume of 270 mL or after 180 minutes, the fermentation volume increases more steeply. After about 300 minutes, 450 mL is reached. After 400 minutes, the fermentation height is approximately 580 mL, and the curve then rises slightly further before stagnating at a value of approximately 600 mL at 600 minutes and remaining unchanged until the end point of the measurement.

[0161] V6 also starts at 270 mL at time 0 minutes and rises to 300 mL after 180 minutes. V6 exhibits the steepest increase of all the curves compared here, reaching 580 mL after 400 minutes, which then increases to 600 mL and remains constant at this value until the end of the experiment.

[0162] While VI appears inactive, V3, V4, V5 and V6 can achieve doubling or almost tripling of the fermentation height and thus the central leavening property of a sourdough.

[0163] Example 5:

[0164] Measurement of viable bacterial counts after 1 week and after 4 weeks in VI and V3 F. sanfranciscensis was mixed as a sourdough starter pure culture with VI or V3 and subsequently frozen at -18°C after one week and after four weeks the viable bacterial counts in CFU / g were determined.

[0165] Table: 1

[0166] As can be seen from Table 1, the results show that with formulation 1 the number of F. sanfranciscensis increased from 1.2*10 9 after one week to 5.0*10 6 reduce after four weeks of frozen storage.

[0167] With formulation 3, the number of F. sanfranciscensis decreases from 7.9*10 8 after one week to 2.3*10 8 after four weeks of frozen storage.

[0168] Likewise, F. sanfranciscensis was mixed with V3, V4, V5 and V6 and the viable counts were determined after freezing and after four weeks of frozen storage at -18°C.

[0169] Table 2:

[0170] As can be seen from Table 2, the results for F. sanfranciscensis show a viable count (CFU / g) after freezing or after four weeks of frozen storage of 7.9*10 8 or 2.3*10 8 for V3 a linseed flour antifreeze composition, 7.7*10 8 or 4.0*108 for V4 an antifreeze composition containing linseed flour and glycerin, 7.1 l*10 8 or 2.9*10 8 for V5 a chia flour GSZ and 6.7*10 8 or 7.1*10 8 for V6 antifreeze composition containing chia flour and glycerin.

[0171] Finally, S. cerevisiae and Lactobacillaceae strains were used for

[0172] Sourdough starter cultures were tested for viable bacterial counts (CFU / g) after freezing, after 3 months, after 6 months, after 3 weeks at -18°C, followed by storage for 5 days at room temperature and refreezing to -18°C.

[0173] Live counts of S. cerevisiae and Lactobacillaceae from fresh sourdough starter culture, directly after freezing, after 3 months of deep-freeze storage, after 6 months of deep-freeze storage and after 3 weeks at -18°C storage, subsequent storage for 5 days at room temperature and then refreezing.

[0174] Table 3:

[0175] As can be seen from Table 3, the results for s, cerevisiae and for Lactobacillaceae (mixed sourdough microbiota) show viable counts (CFU / g) of fresh sourdough starter culture of 2.0*10 7 or for Lactobacillaceae 9.9*10 8 , directly after freezing 6.5*10 6 or for Lactobacillaceae 2.9*10 8 , after 3 months of deep-freeze storage of 3.7*10 5 or for Lactobacillaceae l.2*10 8 , after 6 months of deep-freeze storage of 5.2*10 s or for Lactobacillaceae 8.8*10 7 and after 3 weeks at -18°C storage, subsequent storage for 5 days at room temperature and then refreezing of l,7*10 6 or for Lactobacillaceae 2.1*10 8 .

[0176] Example 6:

[0177] Long-term storage trials with sourdough starter consortium AI (Mmicrobiota A

[0178] 6, A: Influence of GSZ with glycerol

[0179] The sourdough starter was frozen with the GSZ according to the invention and glycerol and analyzed after different storage times (fresh, after 1 week, after 3 months, after 6 months).

[0180] The recipe for the sourdough starter with vegetable GSZ and glycerin was prepared according to the following recipe:

[0181] After mixing to a crumbly consistency, the mixture was allowed to mature at 28°C for 1.5 hours. After the maturation period, the mixture was mixed again to ensure a more even distribution. The mixture was then packaged in plastic containers (200 g each), allowed to mature for 8 hours at 28°C, and then shock-frozen at -30°C.

[0182] Subsequently, the samples were thawed at room temperature (approx. 25°C), stored for 5 days and then refrozen at -18°C.

[0183] The reference was a sourdough starter that was not frozen and to which no glycerin was added. The reference was prepared according to the following recipe:

[0184] After mixing to a crumbly consistency, the dough was left to mature at 28°C for 1.5 hours. After the maturation period, the dough was mixed again for homogenization. The sourdough was packed in 200g plastic containers, allowed to mature for 8 hours at 28°C, and then stored at 4°C.

[0185] 6.B: Acid development after long-term storage tests with sourdough starter A

[0186] Using frozen sourdough starter cultures with microbiota A, metabolic acid development was recorded to demonstrate the reactivation and viability of the sourdoughs.

[0187] For this purpose, homogeneous sourdoughs were prepared from 20 g of “fresh sourdough starter” or frozen sourdough starter, 100 g of wheat flour type 1050 and 110 g of water.

[0188] With these sourdoughs, pH value curves were recorded at 28°C using a pH logger over a period of 20 hours, which recorded the acid development of the test doughs to be compared at different times, namely after 1 week, after 3 months and after 6 months.

[0189] 6.C: Gas volume development after long-term storage tests with sourdough starter In addition, gas volume development was recorded during the fermentation of “fish” and “frozen” sourdough starter after different storage times, namely after 1 week, 3 months and 6 months, in order to measure the metabolic activity and leavening power of the sourdough cultures to be compared.

[0190] The following sourdough starter recipe was used: The gas volume during fermentation was measured. For this purpose, samples of sourdough starter were used "fresh" (reference) and frozen after various storage times according to the above recipe.

[0191] A homogeneous sourdough was prepared and the resulting volume of CO2 in mL / g was recorded in a measurement period of 20 hours of the sample to be compared after 1 week, after 3 months and after 6 months using a gas volume meter at 28°C.

[0192] This experimental setup shows the pH values ​​of the "fresh" reference sample and the corresponding frozen sample at different storage times to measure acid development under suitable fermentation conditions over a period of up to 20 hours (Fig. 3). In Figure 3, the pH measurement of the control sample—fresh sourdough without freezing—starts at a pH of 5.5 at the start time of 0 hours and then decreases sigmoidally to pH 3.7 over the period up to 20 hours.

[0193] The samples frozen with GSZ, which were frozen for one week, began the pH measurement at 0 hours, at a pH of 5.4, which decreased slightly within the first hour before rising again at the same rate as the decrease. The curve then descended sigmoidally until reaching a pH of 3.75 at the end of the test after 20 hours. The sourdough, frozen for three months, started at a pH of 5.95 at 0 hours, then decreased for 7 hours, then continued to develop acid in a sigmoidal curve, finally reaching a pH of 3.8 after 20 hours. The sourdough, which has been frozen for six months, starts after thawing at a comparably high pH value of 5.95 and drops slightly after 7 hours until, surprisingly, the acid development after 20 hours even ends up slightly below the pH value of the three-month frozen cultures at a pH value of 3.7.

[0194] The experiments with sourdough cultures frozen with GSZ show that the cultures and cocultures survived the stress caused by the freezing process better with the GSZ and can be reactivated more quickly and completely to start the desired fermentation processes.

[0195] Example 7:

[0196] Freezing experiments with sourdough starter microbiota C

[0197] Analogous to Example 6, another sourdough microbiota, namely Microbiota C, was tested in the freezing experiments with different antifreeze compositions and after thawing (5 days later) the viable bacterial counts were first analyzed.

[0198] Furthermore, a detailed analysis of the composition was carried out:

[0199] Example 8

[0200] The dough preparation and analysis process was carried out according to the previous examples and at a time point of months after initial freezing. The starter culture is microbiota B.

[0201] Example 9

[0202] Further tests to determine the protection of microbiota A with different amounts of the antifreeze composition according to the invention.

[0203] The dough preparation and analysis process was carried out according to the previous

[0204] Examples and time points days after initial freezing. The starter is microbiota A.

[0205] Example 10:

[0206] Further comparative tests on the use of the GSZ according to the invention in different mixing ratios.

[0207] The dough preparation and analysis process was carried out as described in the previous examples. The starter culture was microbiota A.

[0208] Example 11:

[0209] Further comparative tests on the use of the inventive GSZ in various mixing ratios. The dough preparation and analysis were carried out as described in the previous examples. The starter culture was microbiota A.

Claims

PCT claims 1. Antifreeze composition for single or multiple freezing of microbial cocultures, in particular sourdough, characterized in that the antifreeze composition is free of glycerol and / or DMSO and is composed of: • 5 - 65 wt.% biopolymer and / or biological hydrocolloids, which are selected from the group of ground fenugreek seeds, linseed, chia seeds, psyllium seeds, carob seeds and mixtures of the above, • 7 - 55 wt.% plant and / or microbial mucilage, • 10 - 45 wt% proteins, • 1 - 10 wt% sugar, • 0.1 - 5 wt.% free amino acids, • the vitality values ​​of the frozen cultures normalized within 1-10 hours after thawing.

2. Antifreeze composition according to claim 1, wherein the plant and / or microbial mucilages of the antifreeze composition are water-soluble and are selected from the group containing pentosan, galactan, galactan sulfate, pectin, arabinoxylan, arabinogalactan, glucan, levan, dextran and mixtures thereof.

3. Antifreeze composition according to one of claims 1 or 2, wherein the proteins of the antifreeze composition are selected from the group comprising purified wheat protein, gluten, purified legume protein, pea protein and mixtures thereof.

4. Antifreeze composition according to claims 1 to 3, wherein the sugars of the antifreeze composition are selected from the group of water-soluble monosaccharides and polysaccharides containing galactose, maltose, oligofructose, dextrose, arabinose, trehalose, and mixtures thereof.

5. Antifreeze composition according to one of claims 1 to 4, wherein the free amino acids of the antifreeze composition are selected from the group consisting of proline, glutamates, arginine, alanine and mixtures thereof.

6. Antifreeze composition according to one of claims 1 to 5, wherein the antifreeze composition is selected from the group of flour-like compositions consisting of flour imitations, rice flour, linseed flour, quinoa flour, buckwheat flour, corn flour, chia flour, chickpea flour, lentil flour, pea flour, pomegranate seed flour, coffee bean flour, flour-ground presscakes of oilseeds, namely pumpkin, sunflower, hemp or rapeseed, and mixtures of the above.

7. A process for the freeze-preservation of sourdough, wherein the sourdough is a microbiological coculture of lactobacilli and yeasts, and the process comprises the following steps: Cooling the sourdough to a temperature between 18°-5° C Mixing said sourdough with 10-70 wt.% of an antifreeze composition according to any one of claims 1-6, optionally further cooling, Freeze the mixture at -12°C to -30°C.

8. A process for freeze-preservation according to claim 7, characterized in that it is a two-stage or multi-stage freeze-preservation, wherein after a first thawing of the sourdough, the process steps are repeated for a second or multiple freezing.

9. A method for cryopreservation according to claim 7 or 8, characterized in that the microbial coculture contains lactobacilli selected from the group containing lactobacilli, companilactobacilli, fructilactobacilli, furfurilactobacilli, lacticasebacilli, lactiplantbacilli, lentilactobacilli, levilactobacilli, limosilactobacilli, lactococci, leuconostocci, pediococci, Weissellas and mixtures thereof and further contains yeasts selected from the group containing Kazachstania, Candida, Saccharomyces, Dekkera, Kluyveromyces, Torulaspora, Wickerhamomyces and mixtures thereof.

10. Frozen sourdough produced according to the process of claims 7 to 9, characterized in that the sourdough reaches a gas fermentation with a gas volume development of at least 200 ml up to 600 ml within 1-10 hours after thawing.

11. Frozen sourdough produced according to the process of claims 7 to 9, characterized in that the sourdough achieves an acidification of a starter to pH values ​​of at least 5.5 to 3.5 within 1-10 hours after thawing.

12. Frozen sourdough produced according to the method of claims 7 to 9, characterized in that the thawed microbial coculture contains metabolically active lactobacilli and / or yeasts, wherein the lactobacilli are selected from the group comprising lactobacilli, companilactobacilli, fructilactobacilli, furfurilactobacilli, lacticase bacilli, lactiplant bacilli, lentilactobacilli, levilactobacilli, limosilactobacilli, lactococci, leuconostocci, pediococci, Weissellas and mixtures thereof.

13. Frozen sourdough produced according to the method of claims 7 to 9, characterized in that the thawed microbial coculture contains metabolically active lactobacilli selected from the group consisting of L. acidophilus, L. amylovorus, L. crispatus, L. helveticus, L. johnsonii, Co. alimentarius, Co. crustorum, Co. mindensis, Co. paralimentarius, Fr. fructivorans, Fr. lindneri, Fr. sanfranciscensis, Fu. rossiae, Fu. siliginis, Lc. paracasei, Lp. plantarum, Lp. pentosus, Le. buchneri, Le. parabuchneri, Lv. brevis, Lv. cerevisiae, Lv. hammesii, Li. fermentum, Li. frumenti, Li. mucosae, Li. panis, Li. pontis, Li. reuteri, Li. secoliphilus, L. loctis, L. citreum, P. ocidiloctici, P. porvulus, P. pentosoceus, W. ciborio, W. confuse, W. paramesenteroides and mixtures thereof.

14. Frozen sourdough produced according to the method according to claims 7 to 9, characterized in that the thawed microbial coculture contains metabolically active lactobacilli and / or yeasts, wherein the yeasts are selected from the group comprising Kazachstania humilis, Candida milled, Candida humilis, Kazachstania exigua, Saccharomyces exigua, Dekkera bruxellensis, Kazachstania unispora, Saccharomyces unisporus, Kluyveromyces lactis, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces postorianus, Torulospora delbrueckii, Torulospora pretoriensis, Wickerhamomyces anomalus and mixtures thereof.

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