Production of cake and bread by a Large-scale Aerobic Liquid Culture Starter of New Yeast
Patent Information
- Application Number
- KR1020240008826
- Authority / Receiving Office
- KR · KR
- Patent Type
- Patents
- Current Assignee / Owner
- Filing Date
- 2024-01-19
- Publication Date
- 2026-10-13
- Estimated Expiration
- 2044-01-19
Smart Images
Figure 112024007543650-PAT00029_ABST
Abstract
Description
Technology Field
[0001] The present invention relates to a confectionery and baking method using a large-capacity aerobic liquid culture starter of novel yeast, and more specifically, to a ‘confectionery and baking method using a large-capacity aerobic liquid culture starter of novel yeast’ which is considered to be useful in the field of confectionery and baking. Background Technology
[0002] The natural sourdough starter is derived from domestic rye flour and involves the addition of water to facilitate fermentation. When wheat flour, water, and other additives are subsequently added to form the dough, the microorganisms proliferating within the dough (particularly yeast and lactic acid bacteria) become activated, secreting various extracellular enzymes to drive the fermentation process. After undergoing several expansion cycles, a portion (40–50%) of the sourdough is used for product manufacturing, while the remainder is stored at a low temperature for subsequent fermentation and use in the next expansion.
[0003] Since the existing 'natural sourdough starter' manufacturing process is a non-sterilization technique, contaminants introduced from the moistened rye itself, domestic wheat flour, or the weight-increasing process multiply faster than yeast, which poses a problem. Additionally, after multiple subcultures, there are issues such as the induction of strong off-odors and rapid liquefaction of the dough. Contaminating bacteria ( Bacillus spp. The growth rate of ) is significantly faster than that of yeast, and due to its strong ability to decompose protein sources, it causes off-flavors and inhibits the metabolic activity of yeast growth and fermentation processes. To eliminate these off-flavor-causing microorganisms, pure isolation was performed via diluted plate culture, and a single colony was selected and identified for its rapid rise suitable for baking characteristics. Since yeast derived from such an isolated colony is advantageous in terms of uniformity, a 'large-capacity aerobic liquid culture starter of novel yeast' was devised.
[0004] Sourdough starters are closely related to the proliferation and metabolic activity of the number of living microorganisms (hereinafter omitted), and are closely influenced by the temperature of the water used, the management method, the management period, and the fermentation period; therefore, it is bound to be quite difficult to make the exact same starter every time. Consequently, efforts are required to produce a uniform starter, and it has already been confirmed by inventors and others with experience in using liquid mushroom spawn that the technique of a 'large-volume aerobic liquid culture starter of novel yeast' would be useful.
[0005] In liquid culture techniques, the growth rate of microorganisms must be considered, and the growth rate of microorganisms (2 n , e.g., in the case of bacteria, one generation can multiply every 30 minutes) is fast Bacillus spp. If the genus proliferates relatively better and there is a high-density dominant yeast, the proliferation rate would generally not be as described above; however, assuming theoretically optimal conditions, if one living cell passes after 24 hours, 2 48 The characteristics of microorganisms, which multiply over time to increase to as many as 140 to 280 trillion cells, must be taken into deep consideration. Verification must be performed keeping in mind that a small amount of contaminants coexist with the majority of yeasts, and a method to verify the presence of contamination without fail is already established by using a simple staining method (which can be confirmed within 10 minutes using Giemsa's stain solution) during the optical microscope examination process before use.
[0006] As disclosed in the prior patent, as a result of requesting identification analysis of three yeasts with excellent fermentation ability to the KCTC analysis institution, a novel Saccharomyces cerevisiae ( S. cerevisiae It was identified as a ) strain, and among them, Saccharomyces cerevisiae(. S cerevisiae The GDO Y45 strain was deposited with the KCTC (Korea Conservation Center) on June 26, 2023, and was assigned the accession number KCTC15481BP.
[0007] There are prior Korean patents such as KR1020110120575A, KR1020120077150A, and KR1020120139155A, and Example 6 of KR1020140121028A: setting of a culture method when manufacturing a liquid starter, and a method for manufacturing a sourdough starter in the production of natural fermented bread and food, and there is literature regarding a small-capacity liquid starter system using expensive nutrients.
[0008] To evaluate the inhibition of yeast by alcohol for the leavening of Korean wheat, yeast tolerance was assessed at various alcohol concentrations, and the effects of temperature and nutrients were also examined.
[0009] In this invention, research was conducted to apply a 'large-capacity aerobic liquid culture starter' method to enable commercial use by scaling up the existing triangular small-capacity and medium-capacity manufacturing processes of sourdough starters.
[0010] Korean Registered Patent No. 10-1753372 contains the most similar description, and their aerobic culture method uses expensive reagents such as peptone and yeast. This medium composition generates a high content of organic acids, which causes a decrease in the pH of the medium. It can be predicted that rapid proliferation of the cells leads to a rapid decrease in the number of cells due to the depletion of nutrients, and an increase in the number of dead cells. Additionally, regarding the preparation of liquid spawn for mushroom cultivation, literature (Shim, KK et al. 2012) and experience were referenced, which showed that if the amount of air injected is low, the mycelium is formed at a low density (1.0 kg.f / ㎠) even after a certain period, whereas if the culture medium and mycelium are circulated with a high concentration of strong air pressure (e.g., 2~2.5 kg.f / ㎠), high-density cells are well formed. During air injection, a high air pressure of 1–2.5 kg·f / ㎠ was injected through a three-stage filtration filter, which corresponds to the respiratory reaction equation {a(C6H 12As in {O6)n + bO2-> xCO2+ yH2O + zEnergy}, in the metabolic mechanism, a (nutrient; C6H 12 It is known that O6) and bO2 (oxygen) are required, and that carbon dioxide (CO2), which causes swelling, is produced as a byproduct. However, it must also be considered that if the reaction becomes excessive during this process, high molecular weight molecules are broken down into low molecular weight molecules and saccharification occurs, resulting in a large amount of water (H2O) being produced, which can cause the liquefaction of the dough to proceed easily.
[0011] Considering that if the nutrients used in the metabolic mechanism are abundant in the production of liquid inoculum starters, the mycelium increases rapidly in the logarithmic cycle and organic acids become abundant, resulting in a poor culture environment and a high number of dead cells that may cause off-flavors, the nutrient source should be limited as much as possible and the yeast proliferation rate should be slow, and there should be absolutely no foreign microorganisms other than yeast. Using high-molecular-weight Korean wheat as a liquid culture source is advantageous because it decomposes slowly, and organic acids are generated at a minimum, resulting in minimal pH fluctuations. By maintaining a relatively neutral pH, yeast vitality is not reduced, and the product was manufactured using minimal nutrients and at the lowest cost.
[0012] As revealed in the previous application (Korean Application No. 10-2023-0093815), in a comparison of the 'natural sourdough starter' stage (standard), the triangular small-volume liquid culture stage, the 9L volume liquid culture stage, and the Korean wheat fermentation stage, even if the number of viable cells temporarily decreased in the liquid culture stage, it recovered to a higher number of viable cells in the subsequent Korean wheat fermentation stage compared to the 'natural sourdough starter'. Furthermore, even after baking, in a comparison using sugar-containing and sugar-free liquid culture media in the 9L volume liquid culture process, the product using the latter liquid culture medium (yeast) made only of Korean wheat was preferred. Prior art literature
[0013] Republic of Korea Registered Patent No. 10-2077760 (February 10, 2020) Republic of Korea Registered Patent No. 10-1095805 (December 12, 2011) Republic of Korea Published Patent No. 10-2002-0063163 (August 1, 2002) Republic of Korea Registered Patent No. 10-1753372 (June 27, 2017)
[0014] Shim, Kyu-Kwang et al., 2023, Isolation and identification of yeast derived from natural sourdough starter (Saccharomyces cerevisiae GDO Y45), Journal of Agriculture & Life Sciences Volume 54(2):43-51. The problem to be solved
[0015] S. cerevisiae GDO No. Y45 strain is a yeast that basically adapts well to whole rye and, when adapted well to Korean wheat, has a strong alcohol odor and suitable flavor, and exhibits rapid expansion power in the fermentation of Korean wheat. When fermenting using only Korean wheat, shaking culture was performed for small volumes, and for medium and large volumes, air filtered through a three-stage filtration filter was injected to culture in a liquid state. During the fermentation stage of Korean wheat, fermentation was carried out by adding Korean wheat and 'liquid culture starter' in as equal amounts as possible.
[0016] Previous studies have demonstrated that using selected strains with excellent fermentation activity allows for increased density of pure yeast strains, produces an intense alcoholic odor without off-flavors, and promotes rapid dough rise; therefore, to perfect the technique of a 'large-scale aerobic liquid culture starter of novel yeast' in this study, the liquid culture solution should be used after undergoing a contamination check. Large-scale yeast liquid culture is cultured under aerobic conditions after inoculation, and the presence of contamination can be checked before using the solution. As a method for contamination testing, a staining process using a concentrated Giemsa's stain solution is performed (Shim, KK et al., The contamination check before inoculation at the liquid spawn on Flammulina velutipes. Journal of Mushroom Science and Production. 2012. 10:44-48) The time required is 10 minutes, during which the presence or absence of contamination can be confirmed by examining the specimen with an optical microscope. This is possible because the frequency of subsequent contamination is reduced in specimens in which organic acids and various enzymes are secreted by the dominant bacterial colony. means of solving the problem
[0017] Although yeast strains have a relatively fast growth rate and the characteristic of coexisting and multiplying well even when other types of microorganisms are mixed in, it is best to cultivate and use only the necessary beneficial bacteria without contaminating bacteria that cause off-odors. However, in the 'natural sourdough starter' method using a non-sterilization method, even if the number of off-odor-causing microorganisms is relatively small in the early stages of fermentation dough, if their growth rate is faster than that of yeast, they will multiply increasingly rapidly during the repeated generation process, leading to severe off-odors and rancidity, which necessitates discarding the flour dough and becomes a major cause of reduced product value even after baking.
[0018] In the method disclosed in the aforementioned application filed by the researcher {Application No.: 10-2023-0093815 (Application Date: July 19, 2023)}, when continuously subculturing, mixed heterogeneous bacteria other than yeast strains ( Bacillus spp It was previously confirmed that because the proliferation rate of the . type) was faster, if the subgeneration process was carried out a few more times, the off-flavor became stronger and the fermentation power weakened. The identification of the strains causing major contamination was requested from KCTC.
[0019] The ultimate goal of this study was to improve existing small-volume 'natural sourdough starters' and complete a technique for a 'large-volume aerobic liquid culture starter' using a novel yeast with strong fermentation power. In the production of small volumes, such as in Erlenmeyer flasks, the objective can be achieved without much difficulty by applying aseptic techniques. However, when dealing with large volumes, it becomes necessary to apply methods that are more logically sound regarding the procedures to prevent the introduction of foreign contaminants.
[0020] In particular, various causes of failure in such large-scale spawn production must be examined, including insufficient verification of contamination at different stages of spawn subculturing, inadequate sterilization of large-volume culture media (insufficient time regarding sterilization temperature, pressure, and duration), carelessness during the cooling stage of liquid culture vessels after sterilization, manufacturing defects such as the welding condition of the vessels, poor sealing of the vessels after the addition of nutrients, instantaneous influx of unfiltered air during inoculation, defects in air filters, and the influx of excessive condensate contained in the injected air. Even if yeast possesses the characteristic of coexisting well with other microorganisms, liquid culture that merely establishes dominance while allowing for the presence of minute contamination cannot be used. Just as there was a lack of understanding regarding the problem of minute contamination being induced before aerobic culture is completed by mushroom mycelium—which is much slower than yeast—caution is also required in yeast cultivation.
[0021] While increased capacity offers convenience, failure to detect exposure to contaminants necessitates the disposal of large quantities of material. This can lead to a loss of trust, such as disrupting the rhythm of stable production, in addition to the waste; therefore, it is essential to inspect the cultured liquid for contamination before use in the fermentation of Korean wheat.
[0022] In the baking industry, there was an intention to apply a method suitable for the purpose of not using sugars (e.g., processed sugars such as sucrose) when manufacturing 'natural sourdough starter'. When manufacturing products using a new yeast, all liquid cultures (e.g., Erlenmeyer flask, 9 L capacity, etc.) were cultured using only Korean wheat and natural salt without low molecular weight sugars. Although the number of viable cells decreased significantly during the liquid production, it was evident that the number of viable cells recovered during the subsequent fermentation of Korean wheat, so it was examined whether it was suitable for large-scale aerobic techniques. Effects of the invention
[0023] It has been confirmed that the novel yeast used in the present invention has a good bacterial growth rate even when using Korean wheat as the main ingredient, and has the advantage of achieving maximum rise in 3 to 4 hours to complete the sourdough for product manufacturing in the third fermentation stage of Korean wheat (3rd day of Korean wheat fermentation) by using a 9 L capacity 'aerobic liquid culture starter' and mixing equal amounts of triangular flask culture solution and a 9 L capacity aerobic culture solution without using commercial dry yeast at all.
[0024] As mentioned earlier, we intend to utilize a technique for manufacturing large quantities of this method, which utilizes small volumes of liquid culture solution. When applying such a large-scale liquid starter production method, ensuring uniformity is enhanced as long as the produced liquid culture starter is free from contamination, and it will be highly effective in achieving standardization during the fermented dough stage of Korean wheat. Cases implemented in this regard are described in detail in the drawings and examples.
[0025] It was determined that if a culture medium using only high-molecular-weight Korean wheat and natural salt as nutrients for the 'aerobic liquid culture starter' is used, gradual proliferation will occur without rapid depletion of nutrients, and the yeast density will be maintained without significant difference. To confirm this, the CO2 concentration emitted from 9 L and 150 L volumes was measured, and the viable cell count was verified simultaneously.
[0026] Since sourdough produced by this method is free from microorganisms that cause strong off-flavors and may be present in 'natural sourdough starters,' the number of viable yeast cells is maintained at an appropriate level and fermentation activity is high even in the fermented dough state. As such, it can be stored and used while refrigerated or frozen, making it useful for distribution as frozen dough or dough in a low-temperature state.
[0027] By perfecting and utilizing the technique of a 'large-capacity aerobic liquid culture starter,' stable and uniform fermentation of domestic wheat becomes possible compared to existing methods, thereby maximizing efficiency in expanding subsidiaries and chain stores or exporting technology overseas.
[0028] From the third fermentation process of domestic wheat at the initial stage of sourdough production using this method, the time required for the final dough for bread making each day is only 3 to 4 hours, allowing for sufficient rise, which offers the following advantages: It has the advantage of easily responding to production volume forecasts for the daily demand required for fresh bread making.
[0029] Meanwhile, the baking industry currently tolerates the addition of commercial dry yeast at a weight of 0.1–0.2% to existing 'natural sourdough starter' doughs. However, according to the practical verification by this researcher, when 0.2g of commercial dry yeast is diluted in 10ml of sterile water and cultured on a plate, as much as 10 17 Even in Seung's dilution experiment group, the viable cell count was 3 x 10 20Judging by the concentration of CFU / 0.2g dry yeast, this indicates that a massive number of yeasts are being introduced compared to the present study. However, in dry yeast formulations, such a large number of yeasts must be introduced for logarithmic metabolic mechanisms to occur; adding a larger amount has the disadvantage of causing off-flavors. Even with a large number of yeasts like in dry yeast formulations, it takes a considerable amount of time for enzyme secretion and various activities to occur, and this method is merely a state of leavening caused by the respiration reaction of a massive number of yeasts. Therefore, by utilizing the benefits of metabolites, the appeal of a 'natural sourdough starter' is enhanced, making it healthier and allowing one to experience better flavor. Consequently, by using a liquid culture method as in this study, a third-increase batch of domestic wheat fermented dough is always ready without using commercial dry yeast at all. Furthermore, since the dough rises within 3 to 4 hours to complete the bread-making sourdough, it becomes easier to handle the volume of bread production orders. Brief explanation of the drawing
[0030] Figure 1 is a representative process diagram of the method for manufacturing a medium-to-large volume aerobic liquid culture starter according to the present invention. Figure 2 is a flowchart of the construction of an air supply air conditioning line system for 'manufacturing a large-capacity aerobic liquid culture starter'. Figure 3 is a test tube photograph comparing the cell mass of small size and big size colonies derived from 'natural yeast strain' during the pure isolation process of yeast. Figure 4 shows large-size colonies originating from GDO No. 35–45 during the pure separation process, and each colony is on a dilution plate (10 4 This is a photograph showing that the number of viable cells on a plate of ) appears with almost equal frequency. Figure 5 shows an oval yeast-like appearance in a photograph confirming the morphology of the novel microorganisms cultured in Figures 3 and 4 under a low magnification optical microscope, and is a photograph representing the culture progression stages of the selected strains of this study (slant culture in a test tube, plate culture in a Petri dish, small-volume liquid culture in a triangular container, etc.). Figure 6 shows 10 during the process of isolating the novel yeast GDO No. Yeast strain. 7~8 This is a photograph of an example comparing the difference in growth rates between normal yeast and contaminated microorganisms on a Petri dish plate in the dilution test group, Figure 7 shows the same as (strains No. 25, No. 26, and No. 26) in Figure 6 during the experimental process. Bacillus spp. This is a photograph of the surface condition of Korean wheat dough when using a liquid culture medium with severe contamination. Figure 8 is a comparison photograph of the formation of a network structure by gluten and the state of a damaged network structure during the third fermentation of Korean wheat using an aerobic liquid culture starter of yeast. The photographs in Figs. 9 and 10 show the measured swelling height (cm) over time in an 8.3L transparent container used to evaluate swelling during the third day (third fermentation) of Korean wheat fermentation. Figure 11 is a photograph confirming the fermentation state after adding commercial dry yeast (Saf-instant yeast red, France) at a weight of 0.2% during the fermentation of Korean wheat. Figure 12 shows the evaluation of viable cell counts under identical conditions, isolated for use as a control in this study. Figure 13 shows the confirmation of a 150L large-capacity culture pattern: CO2 concentration measurement during aeration culture and daily bread-making progress. Figure 14 is a graph illustrating the emission concentrations in Table 14. Figure 15 is a photograph of the change in turbidity of the culture medium during the examination of the alcohol tolerance of the new yeast. Figure 16 is a flowchart presenting a method for verifying contaminants when used in large quantities on a commercial scale, and a flowchart explaining Giemsa's stain solution method for contamination testing. Figure 17 shows the change in the side height of the container when fermentation proceeds for 5 hours in the 6th subculture (swelling state after the 3rd day) of Korean wheat by the newly isolated strain of GDO No. Y40. Figure 18 is a photograph showing bread made using a culture medium with or without sugar. Figures 19 and 20 show the results of baking using a large volume of 150L of yeast liquid culture mentioned in Figures 13 and 14, in the form of a baguette. Figure 21 shows the experimental procedure and results of the NJ tree phylogenetic analysis of the identification results of strains selected by novel pure isolation. Figure 22 is the nucleotide sequence of No. Y36, the identification result of a strain selected by novel pure isolation. Figure 23 is the nucleotide sequence of No. Y40, the identification result of a strain selected by novel pure isolation. Figure 24 is the nucleotide sequence of No. Y45, the identification result of a strain selected by novel pure isolation. Specific details for implementing the invention
[0031] In one aspect, the present invention provides a method for mass-culturing yeast derived from Korean wheat, and said method
[0032] A step of introducing yeast and a culture medium into a fermentation device equipped with a high-pressure compressor, an air dryer, a filtration filter, and a pressure regulator; and
[0033] The method comprises the step of obtaining liquid starter culture by aerobically culturing yeast at a temperature of 20 to 22°C for 2 to 3 days in a medium to large volume while injecting clean air containing oxygen into the fermentation device through a sterile filter.
[0034] The culture medium used for cultivation comprises 100 parts by weight of Korean wheat powder, 5 to 15 parts by weight of natural salt, 10 parts by weight of defatted soybean meal, and 3 parts by weight of an antifoaming agent, respectively.
[0035] The above yeast is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) strain GDO No. Y45(KCTC15481BP) or lactic acid bacteria Weissella cibaria It is characterized by being mixed with the (lactic acid bacteria) strain KW1 (KCCM12223P).
[0036] The above liquid inoculum can be produced through medium or large-capacity culture. In the case of medium capacity, it may be 5 to 20 liters, though not limited thereto, and in the case of large capacity, it may be 10 to 1200 liters.
[0037] delete
[0038] For medium volumes, the process can be performed by culturing a small amount of yeast or lactic acid bacteria strains stored in YA slant medium (4℃) (e.g., shaking culture in triangular liquid medium), checking for contamination, and then culturing aerobically for several days.
[0039] In the case of large volumes, yeast or lactic acid bacteria mixed strains produced by medium-volume culture can be cultured for several days on the aforementioned scale using a specialized medium and used as a starter for confectionery and baking.
[0040] The liquid culture starter can be repeated three or more times. For the first step, it is preferable to use 1 to 10 kg of Korean wheat and 1 to 10 kg of liquid starter at 28 to 30°C. For the second step, it is preferable to add 2 to 20 kg of Korean wheat and 2 to 20 kg of liquid starter to the first culture medium and perform the process under the same conditions. For the third step, it is preferable to add 6 to 60 kg of Korean wheat and 6 to 60 kg of liquid starter to the second culture medium and perform the process under the same temperature conditions for 3 to 24 hours.
[0041] 10 to 120 kg of sourdough that has undergone this process can be treated at 30°C for 3 to 4 hours and fed into the confectionery and baking process, or stored at 4°C.
[0042] The confectionery and baking method using the above-mentioned fermentation starter
[0043] a) A step of dividing the dough after primary fermentation of Korean wheat and yeast culture at 30–33℃ for 4–5 hours;
[0044] b) An intermediate fermentation step in which a certain amount of Korean wheat and yeast culture solution is added to the above primary fermentation liquid and intermediate fermented at 30–33℃ for 4–5 hours;
[0045] c) A step of adding Korean wheat and yeast fermentation liquid to the above intermediate fermentation liquid and molding fermenting at 30–33℃ for 3–24 hours;
[0046] d) a step of secondary fermenting the above molded fermented liquid at 30~33℃;
[0047] e) a baking step of baking the secondary fermented liquid at an oven temperature of 190~200℃ for a certain period of time; and
[0048] f) A step of packaging the baked bread in fixed units after cooling; includes
[0049] Hereinafter, the method for culturing starter culture and the method for confectionery and baking according to the present invention will be described in more detail.
[0050] Saccharomyces cerevisiae used in the present invention ( S. cerevisiaeStrains such as GDO No. Y45 did not produce any off-flavor during the fermentation process of Korean wheat, and had the advantage of allowing the time of the manufacturing process to be predicted because they produced a strong alcoholic odor, enhanced the flavor of the bread, and allowed for significantly faster fermentation power such as leavening. Furthermore, since a method with excellent fermentation power that achieves a maximum rise of 2.8 to 3.6 times the base volume within 3 to 4 hours without using any commercial dry yeast can be practically implemented in the bread production field, commercial use becomes possible once the 'large-capacity aerobic liquid culture starter of the novel yeast' is completed.
[0051] In this study, most experiments were conducted using only Korean wheat, natural salt, and antifoaming agents to align with the common belief in the baking industry that "the principle is not to use sugars (e.g., sugar, glucose, etc.)" in the manufacturing process of "natural sourdough starter." Subsequently, if no restrictions are placed on the nutrients input during the liquid culture process, additives can be developed using yeast cultured with nutrient substances such as sugar, peptone, and yeast extract, which are well utilized for metabolic activity.
[0052] The inventors of the present invention have isolated and identified a yeast suitable for Korean wheat sourdough when proceeding with confectionery and bread making using Korean wheat. The existing 'natural sourdough starter' technique begins by adding water to whole rye flour, followed by the addition of Korean wheat and water, and baking is carried out after a fermentation process by microorganisms such as yeast and lactic acid bacteria for 4 to 5 times (5th to 6th day).
[0053] The outer layer of whole rye (Gogu) contains a robust cellulose component, and in addition to yeasts well adapted to this composition, various microbial communities exist. When moisture is added during the bread-making process using our rye flour, the diverse microbial communities and yeasts attached to the outer layer proliferate by breaking down the cellulose components of the layer and the starch inside. Introduced during this process Bacillus spp.It multiplies relatively quickly compared to yeast, and there is a problem in that when subculturing is repeated in fermented wheat dough, the frequency of off-flavor induction and excessive liquefaction progresses increases.
[0054] In the existing method using 'natural sourdough starter,' the amount of dough per unit is limited to a small quantity of only about 2 to 4 kg due to concerns about contamination, which results in significant stress regarding the management of the 'natural sourdough starter.' Thus, the problem of ensuring safety against contaminants must be resolved by improving the manufacturing method of such small-capacity 'natural sourdough starter' and transitioning to large-capacity production.
[0055] As described in the prior patent (Korean Application No. 10-10-2023-0093815), the use of a triangular culture medium and a medium-volume (e.g., 9 L) liquid starter preparation method was proposed to maintain the sourdough technique of Korean wheat. However, since the medium volume at that time was somewhat insufficient for commercial use, we intend to propose a 'large-volume aerobic liquid culture starter using novel yeast' to supplement this. Even if it is feasible on an experimental scale, when proceeding on a large scale, the immediate problem is that safety devices capable of preventing sources of contamination must be addressed.
[0056] Generally, baking yeast is required to generate maximum carbon dioxide (CO2) within a short period of 3 to 4 hours when mixed with dough, and it must be possible to break down the high molecular weight of starch into low molecular weight and to incorporate organic acids, etc., to address these issues.
[0057] To apply the proposed method, microorganisms such as yeast must be isolated in pure form. Briefly, they were isolated in pure form using the dilution plate method during the process of fermenting and increasing the volume of Korean wheat by successively subculturing it 4 to 5 times from whole rye. Among the independent colonies appearing on the plates, those with large colonies were selected. Subsequently, those showing a positive Esculine reaction and a negative protease reaction without a clear halo in casein-containing YA medium were selected. The selection focused on strains that exhibited good swelling and a strong alcohol odor during the fermentation of Korean wheat, and strains with strong alcohol tolerance were preferred.
[0058] When using conventional 'natural yeast starters,' water or distilled water has been added to maintain the moisture of the dough. However, when using an 'aerobic liquid culture starter' utilizing isolated yeast, the culture medium already contains metabolically active substances (products such as enzymes and organic acids) necessary for the fermentation of Korean wheat, and actively proliferating yeast is present, making it suitable for the fermentation of Korean wheat. In the culture medium prepared using this liquid culture technique, only selected novel pure yeast is cultured, allowing for the maintenance of uniformity during the liquid culture management process. In particular, after the first starting process using the aerobic liquid culture starter (e.g., first and second fermentation of Korean wheat), strong leavening occurs repeatedly starting from the third stage (the third day), making it suitable for Korean wheat fermentation (sourdough). Subsequently, the processing time can be shortened, the influx of unwanted bacteria is completely blocked, resulting in almost no off-flavors and the fermentation of bread dough with high flavor.
[0059] In the third batch of Korean wheat, when a new ingredient (an equal amount of Korean wheat and liquid culture starter) was mixed and fermented in an amount twice that of the previous fermentation amount, it showed maximum rise (2.6 to 3.8 times the base volume) after 3 to 4 hours relative to the base volume, and when the fermented dough was stirred after maximum rise to remove the risen gas, the effect of repeated rise of the dough was observed as a result of the respiration reaction by live yeast.
[0060] It has been confirmed that by applying the aforementioned small-scale liquid culture and medium-scale aerobic liquid culture starter methods, it is possible to bake bread without adding separate commercial dry yeast. Therefore, when using this method on a commercial scale, concerns regarding contamination arise, and thus, considering these concerns, a method to verify the presence or absence of contamination was introduced along with the generation of basic data.
[0061] The technique conducted using the 'large-capacity aerobic liquid culture starter of novel yeast' presented in this study was deemed to be useful in the confectionery and baking fields.
[0062] In confectionery and baking using domestic wheat, the cellulose must be broken down more effectively than that of imported foreign flour in order to utilize whole rye and domestic wheat. Additionally, since domestic wheat has a lower gluten (protein source) content compared to imported flour, there is a problem with weak leavening during fermentation; therefore, to obtain yeast suitable for domestic wheat, yeasts well adapted to domestic whole rye and domestic wheat were isolated. For pure isolation, selection was made based on large-sized colonies that did not overlap with adjacent colonies on dilution plates. Strains that showed good discoloration on Esculine-containing YA agar plates, which exhibit a positive reaction to β-glucosidase activity, were selected, and strains that showed no or almost no proteolytic enzyme reaction during the fermentation of domestic wheat, which has a lower protein content than imported wheat, were obtained. The selected colonies obtained through the repetition of this process were confirmed to be oval-shaped morphologically using an optical microscope.
[0063] Among these, GDO Nos. Y36, Y40, and Y45 exhibited a strong alcoholic odor, and the selected strains originated from large-size colonies. Subsequently, using these selected strains, yeast strains with good fermentation activity (experimental groups with rapid leavening) were selected during the fermentation process of Korean wheat. The three final selected strains were submitted to KCTC for identification. In the identification analysis, the yeast strains were identified through phylogenetic tree analysis of LSU rRNA gene sequencing gene sequences (length 1200 bp or longer) Saccharomyces cerevisiae It was sympathized with.
[0064] These strains (Y36, Y40, Y45) respectively S. cerevisiae GDO No. Y36, S. cerevisiae GDO No. Y40, S. cerevisiae It was named GDO No. Y45, and among these S. cerevisiae One strain of GDO No. Y45 was deposited with KCTC and assigned accession number KCTC15481BP.
[0065] Prior to this study, the 'natural sourdough starter' method involved starting with whole rye and repeating the process of increasing the volume of domestic wheat several times. However, this method had problems with quality control, as the frequency of contamination was high, especially during the summer, and it was difficult to maintain uniformity during the manufacturing process. Consequently, it has been difficult for the baking industry to standardize fermented dough when manufacturing flour dough.
[0066] We have emphasized conditions useful for microbial culture and metabolic activity, such as temperature, humidity, pH, and acidity, which are general fermentation conditions for yeast involved in the main fermentation of flour, and it is necessary to confirm that the viable cell count is uniformly maintained through these indicator values.
[0067] As previously confirmed in the preparation of liquid culture starters, the use of limited nutrients during aeration resulted in gradual changes in daily CO2 concentration; this suggested that the increase in viable cell count would be moderate due to gradual proliferation, allowing for the maintenance of uniformity over a longer period. Therefore, the experiment was conducted by preparing a large-capacity aerobic liquid culture starter using high molecular weight Korean wheat and a small amount of natural salt, without using easily decomposed and absorbed sugars (e.g., disaccharides such as sucrose and glucose, monosaccharides, etc.).
[0068] Figure 1 is a representative process flow diagram of the method for manufacturing a medium-to-large volume aerobic liquid culture starter according to the present invention. Conventional 'natural sourdough starters' have been manufactured in small volumes due to concerns about disposal caused by microorganisms that induce off-flavors originating from air, water, or rye husks during the small-volume manufacturing process. To overcome this, yeast with strong fermentation power from Korean wheat is purely isolated and S. cervisiaeIt was named GDO No. Y45, etc. This strain is derived from an isolated colony and has a strong alcoholic odor. In particular, when used with a liquid culture starter, it can repeatedly maintain vigorous leavening starting from the third cycle (the third day), thereby devising a method that enables bread making without using commercial dry yeast. By proceeding with this method, it can be utilized as a technique to implement a large-scale natural sourdough starter method.
[0069] Figure 2 is a layout of an air supply air conditioning line system for 'manufacturing a large-capacity aerobic liquid culture starter'. Specifically, air is compressed in a large-capacity compressor, moisture is removed in an air dryer device that removes moisture condensed in the compressed air in the air supply line, and then passes through a filtration filter device that filters out foreign substances and a pressure regulator that controls the high-pressure air pressure to low pressure, finally supplying air at a constant pressure of 1 to 2.5 kgf / ㎠ to each liquid culture tank.
[0070] Subsequently, each culture vessel set is configured with a liquid culture vessel that amplifies air filtered through three rows of filters into the culture vessel, and the air strongly aerated from the bottom circulates the culture medium while supplying oxygen, and the bubbles generated during culture are defrosted by an antifoaming agent and then discharged from the top.
[0071] (Example)
[0072] The present invention will be described in more detail below based on the following examples. These examples are presented for the purpose of more easily explaining the present invention, and it will be obvious to those skilled in the art that the present invention is not limited by these examples and can be modified by substitutions and equivalent alternative examples within the scope of the technical concept of the present invention.
[0073] Example 1:
[0074] Figure 3 is a photograph of test tubes comparing the biomass of small-size and big-size colonies derived from 'natural yeast strains' during the pure isolation process of yeast. The test tubes were cultured using YA medium with shaking. Even under identical conditions, the growth of yeast varied significantly depending on the size of the isolated colony at the time of isolation. As a key finding of this study, yeast derived from big-size colonies exhibited a superior growth rate during the pure isolation process.
[0075] Example 2:
[0076] Figure 4 shows large-size colonies originating from GDO No. 35~45 Yeast during the pure separation process, and each colony is on a dilution plate (10 4 This is a photograph showing viable cell counts on plates of ) with nearly equal frequency of appearance. Unless otherwise noted in this content, the viable cell count on each Petri dish plate is expressed as CFU / 0.2ml(g).
[0077] Example 3:
[0078] Figure 5 shows an oval yeast shape in a photograph confirming the morphology of the novel microorganisms cultured in Figures 3 and 4 under a low magnification light microscope, and is a photograph of a stage that can represent the culture progression stages of the selected strains of this study (slant culture in a test tube, plate culture in a Petri dish, small volume liquid culture in a triangular container, etc.). After the small volume in a triangular container, the process proceeds to medium volume (e.g., 9 L glass bottle stage), large volume (e.g., 150 L stainless steel stage), etc.
[0079] Example 4:
[0080] Figure 6 shows 10 during the process of isolating the novel yeast GDO No. Yeast strain. 7~8This is a photograph of an example comparing the difference in growth rates between normal yeast and contaminated microorganisms on a Petri dish plate in the dilution experiment group; the contaminating fungi mainly found in existing 'natural sourdough starters' are in the form of rods (photo not shown) as observed under an optical microscope, as in the following example. Bacillus spp It was confirmed as .
[0081] These were submitted to the KCTC analysis laboratory, and the nucleotide homology analysis of the identified bacteria was performed using comparative analysis of the NJ Tree, MP Tree, and ML Tree, and all Bacillus spp. They were identified as (Tables 1, 2, and 3), and their nucleotide sequences have been omitted from this document.
[0082] Example 5:
[0083] Figure 7 shows the same as (strains No. 25, No. 26, and No. 26) in Figure 6 during the experimental process. Bacillus spp. This is a photograph of the surface condition of Korean wheat dough when using a liquid culture medium severely contaminated by these bacteria. They exhibit a strong off-odor, browning accompanied by a sour smell over time, and often become watery due to excessive liquefaction of the fermented Korean wheat dough. Regarding these three types of fungi, the detailed strain names identified through comparative analysis commissioned to the KCTC analysis institution are No. 25; NJ;ML: Bacillus siamensis (KCTC 13613[99.73%], ML: Bacillus siamensis (KCTC 13613{99.73%}, MP; B. amyloliquefaciens (DSM7 [99.46%]), No. 26; Bacillus tenquilensis (KCTC 13622[100%]), No. 27.: NJ; Bacillus pumilus (ATCC 7061[99.93%]) / B. zhangzhouensis (DW5-4[99.93%]), MP; B. zhangzhouensis (DW 5-4[99.93%]) and is illustrated in Table 1.
[0084] As mentioned in the content of the previous patent application (No. 10-2023-0093815, Novel superior yeast purified and identified and well adapted to domestic wheat), these strains exhibit a superior growth rate compared to the novel GDO No. Y33~45 yeast strains mentioned in the previous application document, secrete powerful proteases, and cause off-flavors; furthermore, all of these strains Bacillus spp. It was identified as a strain.
[0085]
[0086]
[0087]
[0088] In Table 1 above, No. 25; NJ;ML: Bacillus siamensis (KCTC 13613[99.73%], ML: Bacillus siamensis (KCTC 13613{99.73%}, MP; B. amyloliquefaciens (DSM7 [99.46%]),
[0089] In Table 2 above, No. 26; Bacillus tenquilensis (KCTC 13622[100%]),
[0090] No. 27 in Table 3 above: NJ; Bacillus pumilus (ATCC 7061[99.93%]) / B. zhangzhouensis (DW5-4[99.93%]), MP; B. zhangzhouensis (DW 5-4[99.93%])
[0091] Such Bacillus spp. Since the strain is an off-flavor causing strain that must not be introduced during the production of natural sourdough starter, the 'large-scale liquid culture starter' was produced using yeast isolated in a state of complete exclusion through purification (e.g., GDO No. Y45).
[0092] Example 6:
[0093] Figure 8 is a comparison photograph of the formation of a network structure by gluten and the state of a damaged network structure during the third fermentation of Korean wheat using an aerobic liquid culture starter of yeast.
[0094] The production of bakery products requires a continuous series of processes, from dough making to fermentation, shaping, and baking, which necessitates time and significant manual labor. Consequently, failure in the fermented dough (e.g., issues with work efficiency due to off-flavors or excessive liquefaction) results in significant losses on the production floor. Gluten is formed during the dough-making process; it is a complex of gliadin and glutenin, proteins formed when water is added to flour to create dough. This gluten forms the basic framework of the bread, and the leavening process—which allows the bread to retain the gas generated during fermentation—is a crucial step in breadmaking; therefore, gluten formation is essential for producing high-quality bread. However, compared to imported wheat, which contains approximately 12–13% gluten, domestic wheat contains a relatively low amount of around 9%, requiring meticulous effort during the leavening process.
[0095] Example 7:
[0096] The photographs in Figures 9 and 10 show the swelling height (cm) over time in an 8.3L transparent container used to evaluate swelling during the fermentation process on the 3rd day (3rd cycle) of Korean wheat fermentation. The yeast culture solution used was applied sequentially according to the number of days of culture during the aerobic culture process in a 9L container. Specifically, A sourdough was added starting from the 2nd day of the 9L culture, and B sourdough was added starting from the 3rd day of the 9L (GDO No. Y35) culture. For the first batch of Korean wheat, 100g of Korean wheat and 100g of culture solution from day 2 of 9L were added and mixed, and fermentation was carried out overnight. For the second batch of Korean wheat, 200g of Korean wheat and 200g of culture solution from day 3 of 9L were added to the dough from the first batch of fermentation and mixed, and fermentation was carried out overnight. For the third batch of Korean wheat, 600g of Korean wheat and 600g of culture solution from day 4 of 9L were added to the dough from the second batch of fermentation. When the dough reached maximum rise, the Korean wheat fermented dough was stirred to remove air bubbles and placed back to the base state. After time passed and the rise reached its maximum, the height of the side (h cm) was measured and recorded.
[0097] Example 8:
[0098] This graph shows the height measured from the side of the container regarding the rise that proceeded as shown in the photographs of Figures 9 and 10 (Tables 4 and 5). It contains the measured rise height (cm) over time in an 8.3L transparent container, which evaluated the rise during the fermentation process on the third day (third time) of Korean wheat fermentation. It was confirmed that when the fermentation of Korean wheat was carried out by adding an equal amount of liquid culture solution to the Korean wheat, the fermentation power became vigorous starting from the third addition (third day). As shown in Table 4, from the third day onwards, the rise is usually repeated every 3 to 4 hours, so it is not necessary to use commercial dry yeast at all for bread making.
[0099]
[0100]
[0101] Example 9:
[0102] Among the graphs (Table 6) showing the height from the side of the container measured for swelling as shown in the photograph of Fig. 10, B sourdough (a graph showing the swelling of Korean wheat fermentation from the 3rd day to the 6th day of use of liquid starter culture).
[0103]
[0104] In the fermentation of Korean wheat using an aerobic culture starter of novel yeast, the volume was small on days 1 and 2, and the yeast's sufficient proliferation and metabolic mechanisms proceeded slowly. However, during the third fermentation process on day 3, there was a two-day logarithmic preparation period, and since the fermentation volume of the Korean wheat reached a suitable point for risen, the fermentation power was vigorous, causing the dough to rise 2.6 to 3.8 times its base volume, and the maximum risen was maintained after an average of 3.4 hours. It was confirmed that after the maximum risen, if the fermented dough was stirred with a spoon to remove gas and returned to a base state, the risen continued repeatedly. At this point, there is no problem in proceeding with the baking process, and commercial dry yeast is not required.
[0105] Example 10:
[0106] Table 7 shows the graph of the temporal change in the swelling height (h cm) at the base state and the subculture of Korean wheat fermentation for 8 days using a 9 L liquid culture that was cultured under aerobic conditions with air introduced during use after being left to stand for about 9 to 10 days following 9 L liquid culture. The swelling was evaluated after stirring at the maximum swelling level when Korean wheat was added or not added until the 9th day of liquid inoculum culture.
[0107]
[0108] The yeast solution used was a 9L volume of standing culture. The composition of the liquid culture consisted of 100g of Korean wheat flour, 100g of brown sugar, 10g of defatted soybean meal, 5g of natural salt, and 3ml of antifoaming agent. For the inoculum in the pre-culture triangular flask, 100ml of the solution was shaken and cultured for one day, and the solution was left standing for several days after culture. The experiment was conducted to evaluate the results when culture was not performed under aerobic conditions and management was poor; during the main experiment, air was supplied again to restore aerobic conditions.
[0109] In this way, the process was carried out (using 0629-Korean wheat fermentation) considering the case where a 9 L volume (0613-culture solution) of yeast culture solution was neglected, and the fermentation period of Korean wheat was maintained until the 9th day. When adding Korean wheat, an equal amount was used, and the process was carried out by excluding an equal amount before adding Korean wheat and then adding an equal amount.
[0110] It was observed that compared to using a culture medium under aerobic conditions, the variability was greater, swelling was poor, and the swelling time was delayed to an average of 4 to 6 hours. The maximum swelling relative to the respective basal volume also showed a significant variability, ranging from 1.7 to 3.9 times. Additionally, when aeration was not performed, there was an alcoholic odor but also a musty smell; compared to using a medium maintained under aerobic conditions, the results were non-uniform, took longer, and showed a large variability in swelling.
[0111] Therefore, for the future use of aerobic liquid culture starters cultured using low-nutrient sources, it is considered useful to use them at a time when yeast is actively growing and to maintain aerobic conditions well.
[0112] Example 11:
[0113] In this study, the data on the number of viable cells at each stage were organized based on natural sourdough starter as shown in Table 8. Although there is a problem with the small number of experiments, the data was summarized as the average value compiled comprehensively to date.
[0114] The stage in which viable cell counts were measured was labeled as 'GDO origin' after isolation and identification from the 'natural sourdough starter' as follows, and was compared by separating into liquid culture in a triangular flask (GDO origin), aerobic liquid culture in 9 L, and the fermentation stage of Korean wheat using the above liquid culture (natural sourdough starter origin, triangular liquid origin, 9 L liquid origin). In addition, the origin of 150 L volume, the presence or absence of sugar in the 9 L volume, and the comparison of the culture temperature (low and high temperature) in the 9 L volume were diagrammed.
[0115]
[0116] Example 12:
[0117] To facilitate understanding of the contents of Table 8, each dilution factor (e.g., 10 5 ~ 10 10 It is illustrated in Table 9 for easy verification. The 'natural sourdough starter' was used as the standard, and strains isolated and identified from it were labeled as GDO-derived and compared at each dilution factor. Compared to the natural sourdough starter, the viable cell count of the yeast in this study shows that growth is generally significantly better.
[0118]
[0119]
[0120]
[0121]
[0122]
[0123]
[0124] As can be confirmed from the liquid culture in an Erlenmeyer flask (GDO-derived), the 9L aerobic liquid culture step, and the comparison of the presence or absence of sugar in the 9L aerobic liquid culture, the dilution factor 10 5 ~10 10 It showed a higher number of viable cells in each stage compared to the 'natural sourdough starter' (the control group of this experiment), and although it showed a somewhat lower frequency in the triangular and 9 L volume liquid cultures, the effect of showing a high frequency of viable cells again in the Korean wheat fermented dough stage was clearly evident. As a result, it can be seen that even without using commercial dry yeast at all, leavening proceeds well and repeatedly in the Korean wheat fermented dough stage.
[0125] Example 13:
[0126] Figure 11 shows the fermentation status confirmed by adding commercial dry yeast (Saf-instant yeast red, France) at a weight of 0.2% during the fermentation of Korean wheat. When compared to the present study, the fermented alcohol odor was strong, but there was a slightly bitter taste. When the commercial dry yeast was purified using the method of this study, no heterogeneous microorganisms appeared, and colonies presumed to be yeast were observed.
[0127] Example 14:
[0128] Figure 12 shows the evaluation of viable cell counts under identical conditions, isolated for use as a control group in this study. In order to improve the existing method used by companies that fermented sourdough by increasing the volume 4 to 7 times from rye to the Korean wheat fermentation stage and then separately adding commercial dry yeast at a ratio of 0.1 to 0.3% during the final bread-making stage, this experiment used only newly isolated yeast cultured in liquid form, and an aerobic liquid culture medium was used instead of water input during the Korean wheat fermentation process in the natural sourdough starter stage.
[0129] 10 of the diluted plates from the isolation derived from commercial dry yeast 15 Four independent colonies from 2x dilution plates in Petri dishes were subcultured onto slant agar in test tubes and stored at low temperature for use, and GDO No. Y45 strain was 10 7 Equally diluted plates were taken and transplanted one loop at a time.
[0130] Example 15:
[0131] For the first treatment concentration, 0.2g of dry yeast was dissolved in 12ml of sterile YA medium, and a dilution plate was performed. Subsequently, a comparative review was conducted under the same conditions as with the novel yeast. As indicated, no foreign contaminants were detected in the commercial dry yeast, and only colonies of a single yeast species were confirmed by microscopic examination. Among the isolated colonies, some were transplanted and stored in slanted test tubes of YA medium. For the first inoculation, 0.7ml of the pre-culture solution was inoculated into 70ml of triangular Korean wheat medium after 1 loop and shaking for 1 day, followed by shaking for 1 day, and the viable cell count was compared with that of GDO No. Y45 strain.
[0132] Diluted plate culture was performed by mixing with commercial 0.024% dry yeast (0.2g / YA media 12ml), and other experiments were conducted in accordance with the methods of this study.
[0133] As shown in Table 15, when the concentration of 0.024% dry yeast (0.2g / YA media 12ml) was used and the mixture was diluted tenfold, it was confirmed that the number of viable commercial dry yeast cells was added at a concentration of thousands to tens of thousands of times (5,000 to 10,000 times) compared to the Y45 strain in the triangular Korean wheat medium. Consequently, a large amount of bubbles were generated and swelling occurred due to the respiration reaction of a large number of yeast cells that awakened all at once within a short period (3 to 4 hours) without prior fermentation.
[0134]
[0135] Example 16:
[0136] The following is an experiment using commercial dry yeast. A triangular culture medium (50 ml) of YA medium was cultured with shaking for one day, and for the second time, it was subcultured in a triangular culture medium (50 ml) of Korean wheat, followed by use in the Korean wheat fermentation stage. Korean wheat fermentation was carried out by adding Korean wheat for the first and third times, and finally, bread baking was performed. At the stage of adding Korean wheat for the third time, 1 g of each sample was taken and diluted plates were performed to compare and examine the viable cell count.
[0137] Table 16 shows the number of viable cells in the 5th consecutively subcultured triangular liquor and the 5th cumulative Korean wheat fermentation stage in the triangular liquor culture stage as viable cell count (CFU / 0.2ml).
[0138]
[0139] When using the cumulative 5-times subculture in small-capacity triangular flasks, there was not a significant difference between commercial dry yeast and the GDO No. Y45 strain of this study; however, the frequency was somewhat lower because the liquid culture medium consisted only of Korean wheat and natural salt. When this triangular culture solution was added to the fermentation of Korean wheat and 1g was diluted and plate-cultured during the fermentation stage, a much higher number of viable cells was observed compared to the liquid culture of the shaking triangular flask. Even during the culture stage of Korean wheat, there was not a significant difference between the commercial dry yeast and the GDO No. Y45 strain of this study. The reason commercial yeast could not be utilized in the same manner as in this study is that at least 3 days are required for the first attempt at fermenting Korean wheat dough. However, after the first 3 days, continuous and repeated leavening occurs, allowing the effects of the natural sourdough starter to be fully expressed, making it possible to bake bread with improved flavor and digestibility.
[0140] Example 17:
[0141] Table 17 is The liquid obtained from the 5th subculture of the triangular flask shaking culture was used, and the viable number of yeast cells (based on CFU / 0.2ml) in the 5th fermentation with Korean wheat input was evaluated and compared with the control group and the novel strain GDO No. Y45. The triangular liquid consisted of 50ml of YA medium or Korean wheat medium, and a 100ml triangular flask was used, and shaking culture was performed at a temperature of 30℃ and 120rpm.
[0142] Since the initial fermentation volume of Korean wheat is small, an 850ml culture bottle was used, and it was replaced with a 1200ml culture bottle for the 5th addition of Korean wheat. For each addition, 50g of Korean wheat was added along with an equal amount of each yeast strain (Control Yeast and GDO No. Y45) during shaking culture. For each yeast strain, 0.5ml of the previous culture solution was inoculated into 50ml of medium and used after 1 day.
[0143]
[0144] The results shown here are the viable cell counts (10) observed on the 2nd and 3rd days after culturing with a 10-fold dilution for each sample, following up to the 5th subculture and the 5th cumulative addition of Korean wheat. 5 ~10 13 ) was counted (CFU / 0.2ml). The experimental results of the stepwise comparison between the yeast strain obtained through a purification process from the control (saf instant yeast red, derived from dried granules of France) and the identified Saccharomyces servibis GDO No. Y45 strain, which exhibits strong fermentation power well adapted to rye-derived and Korean wheat in this study, are as follows.
[0145] The viable cell counts of the Control (saf instant yeast red, derived from dried granules of France) and the strain isolated by pure isolation in this study were compared after 5 subcultures in the shaking culture stage of a triangular flask. There was almost no difference in the 5th fermentation stage of Korean wheat using an equal amount of Korean wheat and a culture medium shaken for 1 day for each of the isolated yeast strains in each subculture.
[0146] In the viable cell counts obtained over several trials, viable cell counts appeared only at relatively low concentrations during the shaking culture stage in the triangular flask, but viable cell counts appeared at the same level even at high concentration dilution ratios during the fermentation stage of Korean wheat, and viable cell counts appeared at a level almost equivalent to the Control (saf instant yeast red, derived from dried granules of France), and the fermentation (volume of swelling; h cm) during the Korean wheat stage was at an almost equivalent level.
[0147] During the fermentation stage of Korean wheat, the Control (Saf Instant Yeast Red, derived from dried granules in France) exhibited a strong bitter taste, whereas the Saccharomyces servibis GDO No. Y45 strain isolated and identified in this study—which demonstrates strong fermentation power well adapted to the rye-derived and Korean wheat strains—showed a strong sour taste.
[0148] During the fermentation stage, Korean wheat was added five times and fermented. After refrigerating at low temperature for two days, each sample was diluted 10-fold (the experimental group that exhibited severe off-odor during the second addition of Korean wheat fermentation had a pH of 5.94). The final confirmed pH was similar, with the Control (Saf instant yeast red, derived from dried granules of France) at 5.18 and the Saccharomyces servibis GDO No. Y45 strain isolated and identified in this study at 5.17. As mentioned earlier, comparable results were observed during the bread baking process using each strain in the Korean wheat fermentation.
[0149] Example 18:
[0150] Table 18 compares the height of the side walls of the container during the fermentation of Korean wheat, in which commercial yeast was separated under the same conditions as the present method and added to the liquid culture medium.
[0151]
[0152] Yeast derived from commercial dry yeast was cultured as isolated colonies on diluted plates under the same conditions as the present method and compared with the novel yeast GDO No. Y45 under the same conditions. The comparison was made at the 5th subculture stage for triangular culture, and the fermentation of Korean wheat was the 5th stage; the accumulated liquid in the triangular flask used here was also from the 5th subculture. As can be seen in Table 18, the rise height during the fermentation of Korean wheat showed comparable fermentation power.
[0153] When commercial dry yeast strains are isolated into isolated colonies and used as a liquid culture in the same way as in this method, baking becomes possible 3 to 4 hours after the third day of fermentation. Therefore, it was found that much faster leavening occurs when using an 'aerobic liquid culture starter'. In evaluations of leavening conducted dozens of times up to the fifth day with Korean wheat, leavening was maintained without any specific issues between the two experimental groups. Thus, the methodology of using a 'large-capacity aerobic liquid culture starter' was deemed appropriate.
[0154] Example 19:
[0155] Table 19 shows the frequency of appearance of typical viable cell counts (CFU / 0.2ml) when using an aerobic liquid culture starter of 9L-2th (GDO No. Y35) that was cultured in the manner of the present study by isolating isolated colonies derived from commercial dry yeast. As summarized in Table 19, the liquid culture from a triangular flask inoculated with 0.7ml of commercial 0.024% dry yeast (0.2g / YA media 12ml) in 70ml of YA medium and shaken for 1 day is shown in the Korean wheat medium inoculated with the liquid culture from the present method.
[0156]
[0157] Reference: 0712-Dough B:: baked sweet potatoes(Remove the skin)-110g+ 613-triangular culture Y35=Korean wheat of natural salt medium 113g: The frequency of occurrence of the normal number of probiotics
[0158] Example 20:
[0159] Table 20 is a photograph and a comparison graph showing the time-dependent CO2 concentration in the respiration reaction due to the growth rate of yeast in medium combinations with and without added sugar (25g sugar and 0g sugar-free) in 9L aerated culture. To measure the CO2 concentration emitted due to the yeast respiration reaction (ppm), a plastic bag was placed over the outlet to collect the relative amount, which was indicated.
[0160] The CO2 concentration emitted during culture was compared using a 9L glass bottle. For the inoculum, 65g of triangular liquid cultured for 1 day with shaking was inoculated into 9L bottles. Air was injected using a Youngblower at a culture temperature of 30–32℃, and the injected air was Pall Co.'s Acro TMThree filters (PN 4250 / 4251) with a 50 Φ 50 mm, 0.2 µm PTFE pore size were connected in series and injected. The relative CO2 concentration (ppm) emitted was measured using a CO2 concentration meter (AQ-9901SD) with a vinyl cover on the 60 ml cotton container side of the outlet.
[0161] 65g of the triangular liquid cultured for 1 day with shaking was inoculated into a 9L volume, and air was injected using a Youngblower at an incubation temperature of 30–32℃; the injected air was Pall Co.'s Acro TM Three filters (PN 4250 / 4251) with a diameter of 50 mm and a 0.2 µm PTFE pore size were connected in series. The emitted CO2 concentration was measured using a CO2 concentration meter (AQ-9901SD) with a vinyl cover placed over the cotton container portion of the 60 ml outlet. The consumption amount (200–500 ml capacity) used for fermentation during measurement was not taken into account.
[0162] As expected, the CO2 concentration emitted from the sugar-free culture medium showed a steady emission rate without a logarithmic increase, but as shown in Table 00, the yeast was multiplying. Although the growth rate of yeast varied depending on the amount of nutrients input and the duration of culture, the yeast cultured in liquid form showed a significantly higher number of viable yeast cells compared to the existing 'natural sourdough starter' when applying the same amount of culture medium used to prepare Korean wheat fermented dough or a slightly reduced amount to control moisture. For this reason, by applying this method, from the third fermentation of Korean wheat onwards, sufficient leavening suitable for bread making can be achieved even by using only the aerobic culture starter of this study, and the effects of a natural sourdough starter can be obtained through the accumulated fermentation process.
[0163]
[0164] Table 20 shows the evaluation of the presence or absence of 25g of sugar in a 9L aerated culture medium (culture medium composition: 100g of Korean wheat, 5g of natural salt, 10g of defatted soybean meal (excluding sediment), and a small amount of antifoamer in 9L of water). The graph shows the relative CO2 concentration (ppm) emitted and the measured CO2 concentration during culture under aerobic conditions in this medium. The CO2 concentration at the outlet under aerobic conditions of a medium volume (e.g., 9L volume) was measured by covering the end of the syringe cotton container, which served as the outlet, with a bag. The experimental results indicate that the number of yeast cells increases vigorously with rapid decomposition in the case of low-molecular-weight sugars, such as sugar, which yeast can utilize well; conversely, it was confirmed through the emitted CO2 concentration that proliferation was slow in the case of sugar-free Korean wheat.
[0165] Example 21:
[0166] Figure 13 shows the confirmation of a 150L large-capacity culture pattern: CO2 concentration measurement during aeration culture and daily bread-making progress. The capacity can be varied from 50 to 1200 L depending on the equipment, and this experiment is an example of a large-capacity operation.
[0167] The following is a case where a large-capacity stainless steel liquid culture tank (Example: 150L capacity) manufactured using a liquid culture system in a mushroom cultivation facility was inoculated and carried out due to the lack of in-house large-capacity plant facilities. If no problems arise after operating with such a capacity, it becomes easy to proceed with tank capacities ranging from 50 to 1200L. However, to prevent damage from contamination as the scale increases, a contamination inspection (Fig. 16) should be performed prior to use; within a short time using an optical microscope Bacillus spp. Since techniques to inspect for contaminants such as the back are available, there is no problem in safely culturing and commercializing them in large quantities.
[0168] Example 22:
[0169] Figure 14 is a graph illustrating the discharge concentrations in Table 21. The conditions were not identical to those in the case of a small capacity (e.g., 9 L glass bottle) due to differences in the ratio of inoculum and the failure to maintain an appropriate indoor temperature at the experimental site. Specifically, the ratio of inoculum was 1 / 15 of that of a standard 9 L glass bottle, the culture temperature was approximately 18–22°C (lower than the usual 30–32°C), and the air injection volume was low (20–25 ml / min) in the 9 L volume culture, whereas in the 150 L volume culture, it was high (2.0 kgf / ㎠). Since it was impossible to predict relative comparison values as the volume increased, accurate indicator values could not be presented. However, despite attempting this transition to a large volume, it was possible to complete a baguette-shaped bread using the homo culture solution prepared in a large volume, as indicated in the bread-making process. Therefore, when applying a methodology using a large-scale liquid culture starter, if the nutrient source added to the culture medium (e.g., peptone, yeast extract, sugar, etc. as a high-nutrient source) is supplemented, there is no problem in devising and presenting a large-scale methodology.
[0170] Even with the addition of the nutrients used in this study (100g of gelatinized Korean wheat and 15g of natural salt, plus 3–5ml of Antiform (Sigma-Aldrich, A-6426) to remove foam), it was possible to bake bread with sufficient rise and a unique sour taste. During the process of injecting air at a level of 2.0 kgf / ㎠, the CO2 concentration measured near the outlet (where the CO2 sensor was placed from the 60ml syringe in the cotton container) was investigated as shown in Figure (Fig. 00), and the value was measured without considering the amount of culture medium used for experimental purposes in the middle. Collecting data to use these emitted CO2 concentration measurements as indicators can be utilized as useful data.
[0171]
[0172] In order to develop various types of additives for commercial use in the future, it is necessary to vary the composition of the nutrients added to the liquid culture medium.
[0173] When using the aerobic liquid starter culture preparation technique presented in this embodiment, the growth rate of yeast varies sensitively depending on temperature, the ratio of medium composition sources, and the rate of air input. Therefore, if the culture stage (optimization) of the starter medium during sourdough production is carried out according to a set standard as presented below, it is possible to quickly reach stabilization. That is, to improve the conventional method of producing sourdough on a small scale, the number of yeast cells was determined by culturing using a large-scale liquid aerobic culture technique, and the number of viable yeast cells and fermentation power at each stage of production were examined. The inoculation amount for each stage was set to 1%, and in the first liquid culture, 1 loop or 1% was inoculated. In the case of a triangular liquid, culture was performed at 30℃ and 120 rpm. For aerobic aeration culture, a Young blower (Youngnam blower 10a) was used in a 9L volume of culture medium. For large volumes, as shown in Figure 2, after constructing the air conditioning system, dehumidified air passing through the incoming dryer can be injected at a pressure of 1.5~2.5 kg.f / ㎠.
[0174] Example 23:
[0175] Table 22 examined the differences in nutrients added to the culture medium under conditions during cultivation. It is a comparison of viable cell counts with and without sugar in a 9L volume using yeast derived from GDO.
[0176] When sugar was added to the nutrient source of the contained medium (100g Korean wheat + 15g natural salt + 3ml other antifoaming agent), a high viable cell count was observed, while in the sugar-free medium, a low viable cell count was observed. However, despite these differences in viable cell counts, during the fermentation stage of Korean wheat using this culture medium, the difference in yeast proliferation based on the presence or absence of sugar in the previous stage was maintained to the extent that there were almost no problems with fermentation.
[0177]
[0178] Example 24:
[0179] Tables 23 and 24 examined the effect of culture temperature during culture (low-temperature culture; 20–22°C and high-temperature culture; 30–32°C). This is a comparison of the number of viable cells according to the effect of culture temperature (low temperature; 20–22°C and high temperature; 30–32°C) in a 9L volume using yeast derived from GDO.
[0180]
[0181]
[0182] Although there is a distinct difference in the number of bacteria proliferated by temperature, the number of viable bacteria in the 9L container cultured at low temperature increased significantly over time. On the other hand, the 9L container cultured at high temperature showed rapid proliferation from the beginning, and the rate of increase over time was not significant. Thus, there was a distinct tendency for the number of viable microorganisms to differ significantly depending on the nutrient source and temperature.
[0183] In the evaluation of the effects of nutrient sources (sugar-containing and sugar-free media) and culture temperatures in Tables 23 and 24 during the 9L liquid culture stage, the yeast count gradually increased in the later stages of aeration culture compared to the early stages in the low-temperature culture group where the dilution ratio of the Petri dish was high. In contrast, in the high-temperature culture group, the yeast count appeared more frequently in the high-dilution ratio from the beginning of aeration culture. However, even though there were slight differences in the viable yeast count during the liquid culture, no significant difference was perceived when the yeast was added in an equal amount to the domestic wheat during the fermentation stage. It was confirmed, however, that in the experimental group using a 9L liquid culture starter cultured at high temperature, the bread had a stronger sour taste, a more pronounced alcoholic odor, and more liquefaction of the fermented dough. If one has a sense of bread-making, it is possible to select the direction of these differences and adjust and utilize them accordingly.
[0184] Example 25:
[0185] Table 25 shows the results of the alcohol tolerance test of the novel yeast. Regarding the alcohol tolerance of the isolated novel yeast, changes in pH, total acidity, and viable cell count during culture in media with varying amounts of ethanol (EtOH, BAKER ANALYZED, >99.9%) were examined. After sterilization, 0, 3, 6, 9, 12, and 15% ethyl ethanol were added to the YA medium, and 15, 12, 9, 6, 3, and 0 ml of sterile water were added sequentially to achieve the same concentration, respectively, to equalize the total concentration. Then, 0.1 ml of the first (pre-triangular static solution) Y45 was inoculated into the mixed culture medium at a 1% volume, after culturing 9 ml of YA (Test Tube; culture temperature 30°C, shaking at 120 rpm) for one day with shaking. The turbidity, viable cell count (CFU / 0.2 ml), pH, and total acidity of the medium were examined on the 2nd and 8th days of culture in the YA medium treated with concentrations of 0, 3, 6, 9, 12, and 15% ethyl ethanol.
[0186]
[0187] Example 26:
[0188] Figure 15 is a photograph showing the change in turbidity of the culture medium during the examination of the alcohol tolerance of the novel yeast. This photograph shows a turbid state where a large amount of yeast is formed and the yeast adapts to the concentration of EtOH contained, indicating an increase in the number of viable cells. Table 26 examines the changes in pH and total acidity values at each concentration following the addition of ethanol.
[0189] The graph in Table 26 and the photograph in Figure 15 examine the changes according to the concentration of ethanol (EtOH) added to the YA medium at each concentration, and are a table and graph showing the changes in pH and turbidity. Since the highest viable cell count was observed at a 3% ethanol concentration, the yeast proliferation rate accelerates at a 3% EtOH concentration, implying that alcohol can be utilized as a nutrient source, and it appears to have tolerance up to a 6% EtOH concentration. This was confirmed to be slightly more tolerance than commercial yeast (data not presented).
[0190]
[0191] Viable cell count and pH were examined on the second day of liquid culture at ethanol concentrations (0%, 3%, 6%, 9%, 12%, 15%). The viable cell count was not affected by ethanol concentrations up to 0%, 3%, and 6%. At a 9% ethanol concentration, turbidity was observed during the later stages of culture (day 8), and no growth occurred at concentrations of 12% and 15%.
[0192] Regarding the change in total acidity (Std. curve standard reagent; based on tartaric acid as the standard reagent), total acidity increased at 3–6% ethanol concentrations compared to when 0% ethanol was present. The highest total acidity was observed at 3% ethanol concentration, reaching 0.01026 (g / L); however, this value is significantly lower compared to alcohol-producing fruit wines, beer, and makgeolli, and the total acidity value in the YA medium used in this experiment was particularly small. At this time, the viable cell count was maintained at ethanol concentrations of 0%–9%, confirming that the novel yeast possesses tolerance up to 9% ethanol. On the second day, the viable cell count remained at similar levels in media containing 0%, 3%, and 6% ethanol, while at 9%, the growth rate was impaired, resulting in slow proliferation. When cultured until the eighth day of shaking culture, yeast proliferation was confirmed through turbidity even at a 9% ethanol concentration. The turbidity of the related data can be confirmed as shown in the photograph in Fig. 16. Therefore, it was determined that for those who prefer a sour taste, it would be useful to filter the liquid culture medium and add it at a level of 3% ethanol.
[0193] Example 27:
[0194] In Table 20, it was not observed that there were significant changes in pH before culture or on the second day of culture in the alcohol tolerance experiment with varying ethanol concentrations. This was attributed to the fact that yeast does not produce large amounts of organic acids, uses low concentrations of alcohol for metabolic activities, and also has the characteristic of producing alcohol after a large amount of yeast has proliferated.
[0195] In Table 21, it was confirmed that the viable cell counts at each concentration were such that no survival was possible at EtOH concentrations of 12% to 15%, as shown in the photograph in Figure 16. Additionally, as shown in Table 21, it was determined that survival was possible in a shaking culture medium containing 0 to 6% concentrations. Through this experiment, it can be inferred that the number of yeast cells cannot multiply indefinitely during the fermentation stage of Korean wheat because they are affected by the ethanol (EtOH) produced by a large number of yeast cells.
[0196]
[0197]
[0198] In most studies, yeast does not break down high molecular weight molecules well, but most have the ability to break down monosaccharides, disaccharides, and trisaccharides (trehalose). In addition, there have been reports that even isolated yeasts, such as McCann's study
[17] , were unable to produce carbon dioxide using starch.
[0199] This is because, considering the time requirements of bakeries where the risen fermentation of bread dough must be completed within 3 to 4 hours, the use of a new yeast aerobic liquid culture starter resulted in minimal risenation in the first and second rises using domestic wheat, etc., but strong expression from the third rise onwards; this is because even if the number of viable microorganisms is high, continuous time is required for the logarithmic characteristics to be expressed.
[0200] Example 28:
[0201] Figure 16 is a flowchart showing a method for verifying contaminants when used in large quantities on a commercial scale.
[0202] In the use of microorganisms, even if exposed to minute contaminants, they are typically organisms too small to be seen with the naked eye, but their growth rate increases exponentially (under optimal conditions, the growth rate of microorganisms is 2 n It is represented as such. When approaching this with this logic, if we consider one generation to be as short as 30 min, Bacillus spp It has the characteristic of being able to multiply by a factor of two every 30 minutes, so after 24 hours, 2 48 It is necessary to consider the characteristic that it can multiply by a certain amount. That is, under optimal conditions, there will be 140 to 280 trillion living cells after one day, but even if conditions are not optimal and multiplication does not reach the mentioned number, there will still be at least 100 million (arbitrary estimate) in 1 ml of culture solution. Since the presence of contaminants on a slide glass plate can be sufficiently confirmed at such a concentration in any case, this method allows for verification within a short period prior to the main inoculation during the long process of mushroom cultivation spawn preparation, ensuring reliable and simple application of the contamination detection method. The process using Giemsa's stain solution is shown in Fig. 16.
[0203] Giemsa stain was devised by the German bacteriologist Gustav Giemsa. The Giemsa stain solution is a reagent for staining cells and is a method of staining biological tissues that classifies white blood cells and red blood cells. It is generally used for staining blood specimens, and if the purpose is based on the morphology of microorganisms, it is a staining technique that can sufficiently distinguish between yeast and other forms. It is a method that allows staining to be completed in just one minute with a single conc. Giemsa's stain solution and can determine the presence of contamination in a total of 10 minutes.
[0204] The detailed steps of this method are described as follows: a step of spotting and attaching a sample onto a cleanly washed slide glass; a step of drying the sample on the slide glass; a step of spotting conc. Giemsa's stain solution; a step of staining the slide glass by maintaining the staining solution for less than 1 minute; a step of placing the dried slide glass on the microscope stage, focusing with a low-magnification objective lens, and immersing it in water at a high magnification of 100x for microscopic examination; and a step of taking a photograph of the specimen examined by an optical microscope. As mentioned, the presence of contamination can be sufficiently determined using only a small amount of cultured liquid prior to use. This method (Shim, KK et al., 2012) has been practically applied in the field to determine the presence of contamination in the preparation of liquid spawn for mushroom cultivation.
[0205] Example 29:
[0206] Figure 17 shows the change in the side height of the container when fermentation proceeds for 5 hours during the 6th subculturing (swelling state after the 3rd day) of Korean wheat using the newly isolated strain of GDO No. Y40. As mentioned, this photograph demonstrates that using this method, when the Korean wheat and culture medium are mixed in equal amounts from the first 3 days onwards, it is suitable for the fermentation of Korean wheat.
[0207] Example 30:
[0208] Figure 18 is a photograph showing bread made using a culture medium with or without sugar. It appeared that adding or not adding sugar did not have much effect on the preparation of the liquid starter culture. This means that although the number of yeast produced may show some difference depending on the nutrient source added during the preparation of the liquid starter culture, this method of liquid yeast culture is useful.
[0209] Example 31:
[0210] The baguette-shaped bread photographs in Figs. 19 and 20 are the results of bread making using the 150 L large-capacity yeast liquid culture mentioned in Figs. 13 and 14. In the results of the baguette-shaped bread photographs made according to the respective recipes of bakers A and B within the company, even when using a 150 L capacity culture starter, the rise of the product was not particularly bad, and it received a good evaluation in terms of flavor.
[0211] Example 32:
[0212] Figures 21 to 24 show the experimental procedure and results of NJ tree phylogenetic analysis of the identification results of strains selected by novel pure isolation.
[0213] A. Experimental strain :
[0214] 1 GDO Y36
[0215] 2 GDO Y40
[0216] 3 GDO Y45
[0217] B. Request Details: LSU rRNA gene sequencing
[0218] C. Experimental Method :
[0219] Identification of yeast by LSU rRNA gene analysis
[0220] - Align the sequence by referring to the secondary structure of ribosomal RNA.
[0221] - Obtain a list of strains ranked by high similarity from the standard strain database.
[0222] - Conduct phylogenetic tree analysis.
[0223] D. Experimental Results
[0224] LSU rRNA gene sequence (length 1200 bp or more)
[0225] As a result of the identification of the selected and submitted samples, strains No. Y36, Y40, and Y45 are all Saccharomyces cerevisiae It was determined to be.
[0226] Strain identification was performed by submitting strains GDO No. Y36, GDO Y40, and GDO Y45 to the Korea Culture Collection Center (KCTC) at the Korea Research Institute of Biotechnology and Bioengineering (KRIBB) for analysis. For all three submitted samples, LSU rRNA gene sequences (length 1200 bp or longer) were analyzed. As a result of the identification of the selected samples, strains GDO No. Y36, Y40, and Y45 were all identified in the NJ Tree similarity comparison analysis. Saccharomyces cerevisiae It was sympathized with.
[0227] Figure 22 is the nucleotide sequence of GDO No. Y36, the identification result of a strain selected by novel pure isolation.
[0228] 6-1. Strain Y36
[0229] - LSU rRNA gene sequence length: 1261 bp
[0230] - The determined base sequence is as shown in Fig. 22.
[0231] Figure 23 is the nucleotide sequence of GDO No. Y40, the identification result of the strain selected by novel pure isolation.
[0232] 6-2. Strain Y40
[0233] - LSU rRNA gene sequence length: 1269 bp
[0234] - The determined base sequence is as shown in Fig. 23.
[0235] Figure 24 is the nucleotide sequence of GDO No. Y45, the identification result of the strain selected by novel pure isolation.
[0236] 6-3. Strain Y45
[0237] - LSU rRNA gene sequence length: 1269 bp
[0238] - The determined base sequence is as shown in Fig. 22.
[0239] Strain GDO No. Y45 is based on the LSU rRNA gene sequence Saccharomyces cerevisiae It showed 100% similarity with (NCBI Blast). Results of phylogenetic tree analysis using 28S rRNA gene information (Clustal X, Mega7) Saccharomyces cerevisiae It showed 99.9% similarity with NRRL Y-12632(T). Based on these results, strain GDO No. Y45 is Saccharomyces cerevisiae It is determined to be a strain belonging to. Accordingly. Saccharomyces cerevisiae Strain GDO No. Y45 was deposited with KCTC and assigned deposit number: KCTC15481BP (Deposit date: 2023.06.26).
[0240] As described above, although the present invention has been explained by limited embodiments and drawings, the present invention is not limited to the above embodiments, and various modifications and variations are possible from this description by those skilled in the art to which the present invention belongs. Accordingly, the concept of the present invention should be understood only by the claims set forth below, and all equivalent or analogous variations thereof shall be considered to fall within the scope of the concept of the present invention.
[0241] 1) The present invention was made as a result of the research titled 'Bread making using a large-capacity liquid culture starter of novel yeast' under the Industry-Academic Joint Research (No. 2023-0241) of Woosuk University.
[0242] 2) Microbial identification analysis was performed at KCTC and supported by the "Advancement of Central Microbial Resource Center of the Ministry of Science and ICT" from National Research Foundation of Korea (NRF) (No. NRF-2021M3H9A1030164).
[0243] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC15481BP Date of Deposit: 2023-06-26
Claims
Claim 1 The method comprises the steps of: introducing yeast and a culture medium into a fermentation device equipped with a high-pressure compressor, an air dryer, a filtration filter, and a pressure regulator; and obtaining a liquid starter culture by aerobically culturing the yeast at a temperature of 20–22°C for 2–3 days in a medium to large volume while injecting clean air containing oxygen into the fermentation device through a sterile filter; wherein the culture medium used for cultivation comprises 100 parts by weight of domestic wheat powder, 5 to 15 parts by weight of natural salt, 10 parts by weight of defatted soybean meal, and 3 parts by weight of an antifoaming agent, respectively, and wherein the yeast is Saccharomyces cerevisiae with accession number KCTC15481BP ( Saccharomyces cerevisiae A method for mass-culturing yeast derived from Korean wheat, characterized by using strain GDO No. Y45, wherein the fermentation device compresses air in a large-capacity compressor, removes moisture from the compressed air in an air dryer device in the air supply line, and then passes through a filtration filter device that filters foreign substances and a pressure regulator that controls the high-pressure air pressure to a low-pressure one, finally supplying air at a constant pressure of 1 to 2.5 kgf / ㎠ to each liquid culture tank. Claim 2 delete Claim 3 delete Claim 4 A method for confectionery and baking characterized by using domestic wheat and a yeast culture solution according to claim 1 as a starter for confectionery and baking.
Citation Information
Patent Citations
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