• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    METHOD OF PREPARING NATURAL FLAVORIZING FLAVOR KAKUMI. The present invention relates to a method for preparing a natural kokumi flavor and, in particular, a method for preparing a natural kokumi flavor using fermented inosine-5'-monophosphate broth (IMP) or a fermented glutamic acid broth, prepared by a two-stage fermentation process, including a first fermentation stage for fungal fermentation and a second fermentation stage for bacterial fermentation, a natural kokumi aroma prepared by the method and a food composition that includes the natural kokumi aroma. Natural kokumi flavors prepared according to the method of the present invention are prepared using natural raw materials, which are therefore harmless and safe for the human body and can be added to foods to produce intense and dense tastes and to improve the aroma of foods.
    公开号:BR112015008228B1
    申请号:R112015008228-9
    申请日:2014-02-25
    公开日:2020-12-01
    发明作者:Sung Hun Lee；So Youn Eom；Jae Seung Park；Eun Seon Oh；Kwang Hee Lee；Suk Min Jang；Dae Ik Kang；Won Dae Chung
    申请人:Cj Cheiljedang Corporation；
    IPC主号:
    专利说明:

    [0001] [001] The present invention relates to a method for preparing a natural kokumi flavor and, in particular, a method for preparing a natural kokumi flavor using fermented inosine-5'-monophosphate broth (IMP) or a fermented broth of glutamic acid, prepared by a two-stage fermentation process, including a first fermentation stage for fungal fermentation and a second fermentation stage for bacterial fermentation, a natural kokumi aroma prepared by the method and a food composition that includes the natural aroma kokumi. Prior art
    [0002] [002] Amino acids and peptides are used as flavoring components. Recently, natural flavoring materials including amino acids and peptides extracted by fermenting vegetable protein sources, such as soy, wheat or corn, with microorganisms such as fungi, bacilli, lactic acid bacteria or yeasts and by the hydrolysis of the product of fermentation, have been developed in several ways.
    [0003] [003] In general, technology has been developed in order to improve the extracted taste components (amino acids and peptides) by increasing the degree of hydrolysis of vegetable protein sources or by increasing price competitiveness, increasing yield in terms of pasta. However, when only vegetable protein sources are used, there is a disadvantage in that there is no nucleic acid component in the resulting flavoring material and, therefore, the umami flavoring intensity of the flavoring material is low. In addition, flavoring materials must contain glutamic acid together with nucleic acids, such as inosine monophosphate (IMF) or guanosine monophosphate (GMP) in order to increase their commercial value.
    [0004] [004] Recently and in order to overcome the gap mentioned above in relation to the hydrolyzate of vegetable protein, materials containing natural nucleic acids, such as yeast extracts, have normally been used. However, yeast extracts can negatively affect the inherent aroma of processed foods, due to a defect in aroma or odor, resulting from their peculiar fermented odor, and their nucleic acid content is limited (maximum content in nucleic acids: 20%). In addition, mixtures of vegetable protein materials with yeast extracts for use in processed foods have low competitiveness compared to conventional flavoring materials, such as monosodium glutamate (MSG), IG nucleic acid or hydrolyzed vegetable protein (HVP).
    [0005] [005] However, IMP together with GMP is a substance widely used as a flavoring food additive. When IMP is used specifically with monosodium glutamate (MSG), it has a significant ability to improve taste. In this way, IMP is an aromatic substance based on nucleic acids that deserves attention as a flavoring substance.
    [0006] [006] Methods for fermenting IMP and GMP for use as flavoring substances include a method of degrading yeast RNA and a method of preparing IMP and GMP in two stages (fermentation and chemical phosphorylation) after preparation of inosine and guanosine. However, in recent years, in several countries, including Europe, standards for natural flavorings have become more stringent and regulations have become stricter, and therefore, aromas that are not made up of exclusively natural components, but which are prepared by carrying out chemical processes or adding additional components are not recognized as natural flavors. For this reason, natural nucleic acid components should be prepared using a method that directly ferments sugar with the bacteria.
    [0007] [007] In these circumstances, the authors of the present invention have made great efforts to prepare a natural kokumi aroma that does not contain any additional components without performing any chemical process and, consequently, have found that when an IMP or fermented glutamic acid broth is used , produced by a two-stage fermentation process, which includes a first fermentation stage for fungal fermentation and a second fermentation stage for bacterial fermentation, a series of efficient flavors can be produced, even when only fermented broth is used for a subsequent reaction, thus completing the present invention. disclosure Technical problem
    [0008] [008] It is the object of the present invention to present a method for preparing a natural kokumi flavor that does not contain additional components, without carrying out any chemical process and using a fermented inosine-5'-monophosphate (IMP) broth or a fermented broth of glutamic acid prepared by a first fermentation stage for fungal fermentation and a second fermentation stage for bacterial fermentation.
    [0009] [009] Another object of the present invention is to present a natural kokumi aroma, prepared by the method above.
    [0010] [010] Yet another object of the present invention is the presentation of a food composition that includes the natural kokumi aroma above. Technical Solution
    [0011] [011] In order to achieve these objects, in one aspect, the present invention presents a method of preparing a natural kokumi flavor, the method including the stages of: (a) fermenting a source of vegetable protein with fungi to obtain a fermented grain broth; (b) fermenting the fermented grain broth with bacteria to prepare a fermented broth of glutamic acid and (c) mixing the fermented grain broth of phase (a) and the fermented broth of phase glutamic acid (b).
    [0012] [012] Furthermore, the present invention provides a method of preparing a natural kokumi flavor, which method comprises a fermentation stage of the fermented grain broth with bacteria to prepare a fermented inosine-5'-monophosphate (IMP) broth in phase (b) and the mixture with the fermented IMP broth in phase (c).
    [0013] [013] Fig. 1 shows a schematic of a method of preparing a neutral natural flavor according to the present invention.
    [0014] [014] Specifically, a fermented broth of glutamic acid, a fermented broth of IMP and a fermented broth of grains are prepared in a first stage of fermentation for fungal fermentation and in a second stage of fermentation for bacterial fermentation and are subject to a third phase in which an appropriate reaction or treatment for each aroma, thus preparing each natural aroma.
    [0015] [015] Thus, the present invention is characterized by a fermented broth of IMP or a fermented broth of glutamic acid, which are used as raw material, being prepared by a fermentation process in two stages, which includes a first stage fermentation for fungal fermentation and a second fermentation phase for bacterial fermentation.
    [0016] [016] The first fermentation phase for fungal fermentation is a phase of producing peptides and amino acids using a protein source. When only the first fermentation stage is carried out for fungal fermentation, large amounts of peptides and amino acids can be produced and, thus, glutamic acid can be produced which can be used as a flavor. However, there is a disadvantage that a nucleic acid substance, such as IMP or guanosine monophosphate (GMP), cannot be produced, which can promote an umami flavor. In addition, there is a problem when a source of vegetable protein is used as it is only subjected to a proteolytic process and, thus, the peptide and amino acid that can be produced depend on the concentration or content of the protein in the source of protein For example, when beans are used, the glutamic acid content in the fermented broth produced will be less than 10% and when using wheat gluten, the glutamic acid content in the fermented broth produced will be less than 15%.
    [0017] [017] However, the second stage of fermentation for bacterial fermentation is a stage of preparation of fermented broths of nucleic acid and glutamic acid. When only the second fermentation stage is carried out for bacterial fermentation, nucleic acid and glutamic acid can be effectively produced. There is, however, a deficiency in that the content of amino acids and peptides is produced in a concentration below 1% and, therefore, the broth fermented with bacteria cannot be used as a flavor, although it still has a good flavor. umami flavor. In other words, there is a deficiency in that, in order to use the fermented broth obtained only by bacterial fermentation, as an aroma, it is necessary to add additional components to the fermented broth so that it can be applied to food.
    [0018] [018] Consequently, the authors of the present invention have developed a method for preparing a natural aroma, while overcoming the deficiency of the fungal fermentation process and the bacterial fermentation process, using only the fermented broth prepared without adding any additional components. and without performing any chemical process.
    [0019] [019] To prepare a fermented broth of nucleic acid and glutamic acid using the two-stage fermentation process, several mineral salts, amino acids and vitamins are needed together with a carbon source and nitrogen source. In particular, in the prior art, yeast extract or hydrolyzed vegetable protein (HVP) was used as the nitrogen source. But in this case, deficiencies were found in which the resulting fermented broth had a defect in aroma or odor and the yield was somewhat low. In addition, the content of flavor components in the resulting culture broth, as well as the overall flavor, differs greatly depending on the various materials that are used for bacterial fermentation. Thus, in the present invention, a fermented grain broth (grain protein hydrolyzate), which is obtained by fungal fermentation (first fermentation stage) and which can contain several amino acids and peptides at the same time that it serves as a nitrogen source , is used as a substrate for the second fermentation stage for bacterial fermentation.
    [0020] [020] In conventional processes for preparing nucleic acid or MSG using bacteria, particular importance was attached to increasing only the concentrations of nucleic acid and MSG in culture broths and their yields. This increase was effective in increasing the umami flavor, but in some cases, this increase negatively affected the aroma of the resulting flavoring substances. However, the various natural flavors prepared using the 3 phases of the present invention have an advantage in that the aroma can be improved by controlling the concentrations of IMP and glutamic acid, which are components of umami flavor, as well as the various amino acids , saccharides, organic acids and inorganic ions, etc.
    [0021] [021] We now proceed to describe each phase of this method of the invention to prepare a natural aroma.
    [0022] [022] Step (a) of the method of the present invention is a step of fermenting a source of vegetable protein with fungi to obtain a fermented grain broth.
    [0023] [023] In step (a), fungal fermentation is carried out using a source of vegetable protein, thus obtaining a fermented grain broth that contains various amino acids and peptides and includes components, such as glutamine, which can be used as a nitrogen source in the second fermentation stage for bacterial fermentation. Specifically, fungi are cultured using grain material as a substrate to prepare a cell culture broth containing protease, which is then added to a vegetable protein source, followed by hydrolysis, thereby preparing a protein hydroxide of grains. Thereafter, the grain protein hydrolyzate is filtered and the cells are removed from it, thereby preparing a fermented grain broth. The grain protein hydrolyzate may contain a total nitrogen content of 2% (w / v) or more in order to provide a nitrogen source for the second fermentation stage for bacterial fermentation.
    [0024] [024] As a source of vegetable protein, any material known in the art can be used in the present invention, as long as it can be fermented with fungi. Examples of vegetable protein sources include, but are not limited to, soy, corn, rice, wheat gluten, etc. However, when wheat gluten is used, it can advantageously increase the yield of bacterial fermentation since it contains a large amount of glutamine. In this way, wheat gluten can preferably be used as a source of vegetable protein.
    [0025] [025] As fungi, any fungus can be used in the present invention, as long as they can ferment the vegetable protein source to prepare the fermented grain broth of the present invention. The fungi that are used in the present invention can preferably be microorganisms Aspergillus sp. and more preferably microorganisms Aspergillus orizae or Aspergillusoyae, but not limited to these examples.
    [0026] [026] In an example of the present invention, the first fermentation stage for fungal fermentation was carried out using Aspergillusoyae CJCC_080124P (KCCM11026P) as described in Korean Patent Registration No. 10-1191010 (corresponding to PCT International Publication No. WO 2011- 046249).
    [0027] [027] As used today, the term "fermented grain broth" refers to a product obtained by fermenting a source of vegetable protein (grains) with: fungi. The fermented grain broth can be used as a substrate for the second stage of bacterial fermentation and can also be used in the final stage of preparing an aroma by subjecting the fermented grain broth to filtration and cell removal processes after fermentation. In this way, the fermented grain broth can be used for two purposes, as described above.
    [0028] [028] Stage (b) is a fermentation stage of the fermented grain broth, obtained in stage (a), with bacteria to prepare a fermented broth of glutamic acid. Step (b) may further include a fermentation step of the fermented grain broth with bacteria to prepare a fermented IMP broth. Specifically, the fermented grain broth obtained in the first fermentation stage for fungal fermentation is used as a substrate for bacterial fermentation and the IMP fermented broth and the glutamic acid fermented broth can be prepared by subjecting the fermented grain broth to bacterial fermentation in a medium supplemented with a carbon source.
    [0029] [029] Bacterial fermentation can be carried out by a general method of bacterial culture known in the art and preferably can consist of three phases: flask culture, industrial scale culture and main culture. Specifically, flask culture and expansion culture are performed using a primary culture medium and a secondary culture medium in order to obtain a scale, after which bacterial fermentation is performed using the fermented grain broth of the phase ( a) as a substrate in the main culture medium while continuing to supply additional sugar continuously, thereby obtaining a fermented IMP broth and a fermented glutamic acid broth.
    [0030] [030] The medium for bacterial fermentation may include a carbon source such as glucose, fructose and the like. In particular, the means for preparing the fermented glutamic acid broth may include raw sugar as a carbon source. The medium can include various mineral salts, vitamins, amino acids and the like and the composition of the medium can vary depending on the desired fermentation product which is the fermented IMP broth or the fermented glutamic acid broth.
    [0031] [031] For example, when the fermented glutamic acid broth is to be prepared, the main culture medium may include glucose, fructose, raw sugar, betaine, magnesium sulfate, potassium phosphate and phosphoric acid and, preferably, may include, based on the total weight of the medium, 0.5-0.7% by weight of glucose, 0.9-1.1% by weight of fructose, 4.5-5.5% by weight of raw sugar, 0.005 -0.015% by weight of betaine, 0.3-0.5% by weight of magnesium sulfate, 0.8-1.0% by weight of potassium phosphate and 0.2-0.4% by weight of acid phosphoric. In addition, when the fermented IMP broth is to be prepared, the main culture medium may include glucose, fructose, magnesium sulfate, phosphoric acid, potassium hydroxide and the fermented grain broth. Preferably, the main culture medium may include, based on the total volume of the medium, 4.4-5.2% by weight of glucose, 3.7-4.3% by weight of fructose, 1.3-1, 7% by weight of magnesium sulfate, 2.0-2.4% by weight of phosphoric acid, 1.4-1.8% by weight of potassium hydroxide and 0.5-0.9% by weight of the broth fermented grain.
    [0032] [032] Furthermore, the medium may, if necessary, include small amounts of other components, for example, iron sulphate, manganese sulphate, copper sulphate, zinc sulphate, CAPA, NCA (nicotinamide), biotin, chloride calcium, thiamine, vitamin C and the like. Typical examples of each of the media that include these components are shown in Tables 5 to 8 and 9 to 12.
    [0033] [033] As a result of the fungal fermentation in step (a), the plant protein source is broken down into amino acids and peptides and inorganic ions such as calcium, phosphate magnesium ions and vitamins are eluted from the protein source. The amino acids produced by fungal fermentation include glutamine, methionine cysteine, valine, leucine, isoleucine and the like and can be used as a nitrogen source in bacterial fermentation in step (b). In particular, a high concentration of glutamine is an essential component for purine biosynthesis and can act as a major promoter in the production of high levels of IMP and glutamic acid. Furthermore, eluted inorganic ions and vitamins can be useful in the growth of bacterial cells in bacterial fermentation. In an example of the present invention, it was demonstrated that when the product obtained by bacterial fermentation was used as a nutrient source, the proliferation and growth of bacterial cells became faster (FIG. 4). This further demonstrates the advantage of the two-stage process of the present invention.
    [0034] [034] As currently used, the term "bacterium" refers to any bacterium that can ferment the fermented grain broth obtained in step (a) to produce fermented IMP broth and fermented glutamic acid broth. To prepare a natural flavor according to the present invention, a non-GMO strain can be used. The bacteria that are used in the present invention can be any bacterium known in the art, which is capable of producing IMP and glutamic acid by fermentation. For example, when preparing a fermented IMP broth, microorganisms from Bacillus sp., Corynebacterium sp. or Escherichia sp. and when a fermented broth of glutamic acid is prepared, microorganisms of Corynebacterium sp., Microbacterium sp., Bacillus sp., Streptomyces sp., Penicillium sp., Pseudomonas sp., Arthrobacter sp., Candida sp., Klebsiella sp., Erwinia sp., Pantoea sp. or Enterobacter sp. More preferably, the bacteria that are used in the present invention can be microorganisms of Corynebacteríum sp. More preferably, Corynebacterium ammoniagenes can be used to prepare the fermented IMP broth, and Corynebacterium glutamicum can be used to prepare the fermented glutamic acid broth. In addition, as bacteria, various bacteria disclosed in previous patent documents can be used and are known for their ability to produce IMP and glutamic acid. For example, fermented IMP broth can be prepared by the bacterium Bacillus sp. or Escherichia sp. disclosed in Korean Patent Open Publication No. 10-2007-000507 (corresponding to PCT International Patent Publication No. WO 2005-095627), and fermented glutamic acid broth can be prepared using Enterobacter sp. or Klebsiella sp. disclosed in Korean Patent Open Publication No. 10-2000-0029174 (which corresponds to U.S. Patent No. 7247459), without limitation.
    [0035] [035] In an example of the present invention, to prepare a fermented OMP broth, Corynebacterium ammoniagenes CJIP009 (KCCM-10226) described in Korean Patent Registration No. 10-0397321 (which corresponds to International Patent Publication WO N ° WO2002-051984) and to prepare a fermented glutamic acid broth, Corynebacterium glutamicum (Brevibacterium lactofermentum) CJ971010 (KFCC 11039) described in Korean Patent Registration No. 10-0264740 was used.
    [0036] [036] The fermented broth obtained by bacterial fermentation in step (b) has a solids content of approximately 150 g / L. The present invention is characterized in that the fermented broth of IMP and / or glutamic acid obtained by bacterial fermentation is used in a process of preparation of natural flavor without adding any additional component to it, the fermented broth must contain large amounts of IMP and acid glutamic which are the desired components.
    [0037] [037] Thus, solids in the fermented IMP broth prepared by the first fermentation stage for fungal fermentation and the second fermentation stage for bacterial fermentation can preferably contain an IMP content of 30% or more, and more preferably 50% or more, this being no limitation. Consequently, the concentration of IMP in the fermented IMP broth may preferably be 50 g / L to 150 g / L and, more preferably 70 g / L to 130 g / L.
    [0038] [038] Furthermore, since the product produced by the first fermentation phase for fungal fermentation contains amino acids, glutamic acid can be obtained at high concentration and high yield compared to those of IMP. The solid in the fermented broth of prepared glutamic acid may preferably have a glutamic acid content of 50% or more and, more preferably, 60% or more, and this is not a limitation. Thus, the concentration of glutamic acid in the fermented glutamic acid broth may preferably be 75 g / L to 150 g / L and, more preferably 90 g / L to 130 g / L,
    [0039] [039] Due to the high content of IMP and glutamic acid in the fermented broth, as described above, when the fermented broth is enriched by a process such as drying, it can be properly added to the food.
    [0040] [040] As currently used, the term "aroma" refers to a substance that is added to improve the aroma of the food. The aroma can be classified according to its component into several aromas Specific examples of the favor include a neutral aroma, a meat aroma, a chicken aroma, a pig aroma and a kokumi aroma. Each of the flavors can be prepared using a fermented IMP broth and fermented glutamic acid broth by the two-stage fermentation process of the present invention. Among these, the term "kokumi aroma" refers to a substance with kokumi (Japanese) that indicates a rich flavor, an intense flavor, a flavor that fills the mouth, a dense flavor or a sticky flavor. In Britain and the United States, kokumi is used to denote the feeling of a full mouth, continuity, density or pleasure. The kokumi aroma can improve the physical sensation of a product and also the effect of improving the perception of salty.
    [0041] [041] Since the fermented broth of the present invention is characterized by being used in a process of preparation of natural flavor without adding additional components and without subjecting them to an additional chemical process, such as purification, all components of the medium must be food-grade materials so that the fermented broth is directly included in the food, for example, processed foods. In this way, all components of the medium that are added during fermentation are preferably food-grade materials. In an example of the present invention, for edible materials to be used as components of the medium, β-alanine has been replaced by calcium pantothenate (CAPA) and meanwhile, it has been demonstrated that the fermented IMP broth with a concentration of 70 g / L or more could be prepared. Thus, the bacterial fermentation medium according to the present invention can preferably contain CAPA.
    [0042] [042] Each IMP fermented broth and glutamic acid fermented broth can be prepared by the two-stage fermentation process, including stages (a) and (b) and the prepared fermented broth can finally be subjected to a third stage for reaction and can be used in a process of preparing various flavors. Based on the fermented broth of IMP and fermented broth of glutamic acid, various natural flavors, for example, neutral flavors and flavors for meat, chicken, pork or kokumi and the like, can be prepared using different raw materials or slightly altering the composition of the medium or controlling the processing conditions, including temperature, pressure and time, in the process of mixing the fermented broths, or a reaction or electrodialysis process. Among the prepared natural flavors, an aroma appropriate to the purpose of each food can be added to obtain an ideal taste.
    [0043] [043] Phase (d) is a phase of mixing the fermented grain broth and the fermented glutamic acid broth obtained in the previous phases in order to prepare natural kokumi flavors. Furthermore, step (c) may further include a mixing phase with the fermented IMP broth.
    [0044] [044] In step (c), no additional components are added to the fermented broths, the fermented broths are not subjected to an additional chemical process and the fermented broths are mixed together and then reacted under appropriate conditions, including temperature, pressure and time, according to the intended use of the desired aroma, thus preparing natural kokumi aromas.
    [0045] [045] In step (c) the natural kokumi flavors can finally be prepared by mixing the fermented grain broth and the fermented glutamic acid broth using the preparation method of the present invention. In this, the mixing ratio of the fermented grain broth and the fermented glutamic acid broth may preferably be 1: 0.1 to 1:10, more preferably 1: 0.2 to 1: 5 and even more preferably 1: 0.5 to 1: 2.5, which is not a limitation.
    [0046] [046] In addition, the mixed fermented broth can also be mixed with the fermented IMP broth. In this, the mixing ratio of the fermented glutamic acid broth and the fermented IMP broth may preferably be 1: 0.1 to 1:10, more preferably 1: 0.2 to 1: 5 and even more preferably 1: 0, 5 to 1: 2.5. In other words, a natural kokumi flavor of the present invention can be prepared by mixing the fermented broth, obtained through a second fermentation for bacterial fermentation, and the fermented grain broth in an appropriate proportion.
    [0047] [047] After mixing each of the fermented broths as indicated, perfect kokumi aromas can be prepared by reacting between 70 ° C and 100 ° C, preferably between 80 ° C and 90 ° C, preferably for 0.5 to 24 hours, more preferably 1 to 3 hours.
    [0048] [048] However, fermented broths can be used after the filtration process. Therefore, the method of the present invention can comprise a step of treating fermented broths with activated carbon before step (c). In addition, the method of the invention may comprise, after the treatment phase of the fermented broth with activated carbon, a phase of centrifugation or filtration of the fermented broth. After the fermented broth is treated with activated carbon, it can also be treated with diatomaceous earth as an aid in filtration. Furthermore, before the fermented broth is treated with activated carbon, it can be subjected to a pre-treatment heating process of the fermented broth to induce cell lysis, in order to increase the performance of the filtration process that is subsequently carried out. The heating process can preferably be carried out at 70-90 ° C and the heating time can preferably be 15 minutes or more, and more preferably 15-60 minutes.
    [0049] [049] The mixing phase of the present invention can further include a phase of concentrating the fermented broth and drying the concentrate to prepare the powder. The preparation phase of the fermented powder broth can be carried out before or after mixing the fermented broths and can preferably be carried out before mixing the fermented broths. Drying can preferably be achieved by spraying or vacuum drying. The fermented broth can finally be prepared in powder or paste that is suitable for addition to the food.
    [0050] [050] In another aspect, the present invention features a natural kokumi flavor prepared by the method of preparing the present invention. In yet another aspect, the present invention also features a food composition that includes the natural kokumi aroma.
    [0051] [051] The natural kokumi aroma prepared by the preparation method of the present invention has kokumi, that is, a deep physical sensation of the product and a dense flavor and, therefore, can be added to a suitable food to maximize the taste of the food and can also be applied to animal feed. For example, the natural kokumi aroma can be added to soybean paste, bean paste, pasta soup, curry and similar products to produce kokumi. Advantageous Effects
    [0052] [052] Natural kokumi flavors prepared according to the method of the present invention are prepared using natural raw materials, which are therefore harmless and safe for the human body and can be added to foods to produce intense and dense tastes and to improve the aroma of food. Description of Drawings
    [0053] [053] Fig. 1 is a schematic view showing a general process for preparing a natural aroma.
    [0054] [054] Fig. 2 shows an acid hydrolyzing process to obtain MAP in order to replace adenine with a natural food grade material according to the present invention.
    [0055] [055] Fig. 3 shows a metabolic pathway for CAPA derivation, to replace β-alanine with a natural food grade material.
    [0056] [056] Fig. 4 shows the degree of proliferation of bacterial cells in the case of the addition of a degraded vegetable protein by fungal fermentation according to the present invention as a source of nutrients and in case the vegetable protein is not added. Invention mode
    [0057] [057] Hereinafter, the present invention will be described in more detail with reference to examples. However, it will be apparent to those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Example 1: Fungal fermentation from a source of vegetable protein
    [0058] [058] For a primary fungal fermentation, a substrate was grown including grain materials, such as soybeans, corn, rice or wheat, with microorganisms Aspergillus sp. as a fungal strain at a temperature between 20-35 ° C for 24-72 hours, thus preparing a fungal culture broth containing a high concentration of protease. Then, a vegetable protein source, such as soy, corn, rice or wheat, was mixed with water until it reached a high concentration of 25-35% and sterilized, and the phonic culture broth previously prepared and containing a high Protease concentration was added to the sterile vegetable protein source in a salt-free state to hydrolyze the vegetable protein source. The phonic culture broth was added in an amount of 10-100% based on the sterile substrate solution and the substrate was degraded at 40-50 ° C for 48-96 hours to prepare a grain protein hydrolyzate.
    [0059] [059] Specifically, flask and expansion culture were performed using Aspergillus soybean CJCC 080124P (KCCM11026P) as described in Korean Patent Registration No. 10-1191010 (corresponding to PCT International Publication No. WO 2011- 046249) and then the phonic culture broth was prepared using defatted soybean and wheat gluten was hydrolyzed as a substrate, thus preparing a grain protein hydrolyzate. More specifically, 200 ml of main culture was dispensed and sterilized in a 1 L flask and 200 ml of phonic cells were inoculated in the flask at a density of 1.7 x 10 10 ph cells / 400 ml and cultured at 30 ° C and at 100 rpm for 7 hours. Then, a secondary culture was added to a 250 L fermenter and sterilized, and this 600 ml of main culture broth was inoculated and cultured at 30 DC and 70 rpm for 24 hours. Then, a main culture medium was added to a 5-ton preparatory tank and heated at 90 ° C for 30 minutes, after which it was transferred to an 8-ton fermenter and 100 L of water were added and 2 L of a defoaming agent. Then 144 L of secondary culture broth was inoculated into the main culture medium, which was then grown at 30 ° C and 700 rpm for 48 hours, thus preparing a phonic culture broth. Finally, a raw material for hydrolysis of the substrate was added to a 5-ton preparatory tank and heated at 55 ° C for 1 hour, after which it was transferred to a 20-ton fermenter and 100 L of water and 2 L of a defoaming agent, followed by sterilization. Then, 5760 L of a phonic culture broth were inoculated into the substrate and then cultivated at 45 ° C and 30 rpm for 96 hours, thus preparing a grain protein hydrolyzate. The compositions of the media used in the preparation of the grain protein hydrolyzate are shown in Tables 1 to 4 below.
    [0060] [060] The grain protein hydrolyzate prepared as previously described was filtered through a filter press to remove phonic cells, thus preparing a fermented grain broth with a total nitrogen content of 2% (w / v) or more and one degree of hydrolysis of 50% or higher. However, fermented grain broth was prepared as a secondary bacterial fermentation medium. Example 2: Bacterial fermentation
    [0061] [061] To prepare a fermented IMP broth and a fermented glutamic acid broth using fermented grain broth, a carbon source, such as glucose or fructose, mineral salts such as Fe, Mg, Mn and Zn and vitamins to the fermented grain broth prepared in example 1, followed by sterilization, thus preparing the means for bacterial fermentation. The means for preparing an IMP fermented broth and a glutamic acid fermented broth were prepared and, for each of the fermented broths, mediums for flask culture (primary culture), expansion culture (secondary culture) and main culture were prepared. 2-1: Preparation of IMP fermented broth
    [0062] [062] Using Corynebacterium ammoniagenes CJIP009 (KCCM-10226) described in Korean Patent Registration No. 10-0397321 (corresponding to WO International Patent Publication No. W02002-051984) a fermented broth with an IMP concentration of 70 g / L or more.
    [0063] [063] Specifically, 50 ml of a pH-adjusted primary culture with No was dispensed in a 500 ml flask and sterilized at 121 ° C for 15 minutes, then the bacterial strain was inoculated into the medium and grown at 32 ° C and 200 rpm for 22-28 hours. Then 2.1 L of a secondary culture was dispensed in a 5 L jar, sterilized and cooled, after which it was inoculated in this 300 ml of the primary culture broth, having been grown in 2 L of air at 32 ° C and 900 rpm for 27-30 hours, while adjusting the pH to 7.2. Then 8.5 L of the main culture medium was dispensed in a 30 L jar, sterilized and cooled, after which it was inoculated in this 1500 ml of the secondary culture broth and in 5 L of air at 32 ° C and 400 rpm for 5-6 days, while adjusting the pH to 7.2 and providing additional sugar, comprising a mixture of glucose and fructose at any time. After supplying the additional sugar, the culture was carried out for 7 hours or more, in order to allow the total consumption of sugar. The compositions of the media used in the preparation of the fermented IMP broth are shown in Tables 5 to 8 below.
    [0064] [064] Using Corynebacterium glutamicum (Brevibacterium lactofermentum) CJ971010 (KFCC 11039) described in Korean Patent Registration No. 10-0264740, a fermented glutamic acid broth with a glutamic acid concentration of 90 g / L or higher.
    [0065] [065] Specifically, 30 ml of a primary culture was dispensed in a 250 ml flask and sterilized and the bacterial strain was inoculated into the medium and cultured for 5-8 hours. Afterwards 1.4 L of a secondary culture were dispensed in a 5 L jar, sterilized and cooled, and then 20 ml of the primary culture broth were inoculated in this, having been cultivated for 20-28 hours. Then 9.2 L of the main culture medium was dispensed in a 30 L jar, sterilized and cooled, and then in this 800 ml of the secondary culture broth was inoculated, having grown for 36-45 hours with a supply of additional sugar at any time.
    [0066] [066] After supplying the additional sugar, the culture was carried out for 7 hours or more, in order to allow the total consumption of sugar. The compositions of the media used in the preparation of the fermented glutamic acid broth are shown in Tables 9 to 12 below.
    [0067] [067] To prepare the fermented IMP broth using the means of preparation of the fermented IMP broth and to use the fermented IMP broth as a flavor to add to the food, several components of the medium must be replaced by food-grade materials, in order to meet the requirements of a food additive. In this way, adenine and β-alanine necessary for cell growth were replaced by food-grade materials.
    [0068] [068] First, adenine was prepared by acid hydrolysation of AMP, to cleave a β-Ν-glycosidic bond with ribose, in order to release adenine in this way. It has been shown that, when culturing using adenine prepared as described above, it was possible to prepare a fermented IMP broth with an IMP concentration of 70 g / L or more (FIG. 2).
    [0069] [069] Then, as a result of examining the metabolic pathway of Corynebacterium ammoniagenes, it was predicted that β-alanine could be replaced by calcium pantothenate (CAPA). Thus, culture was performed using CAPA instead of β-alanine and, as a result, it was demonstrated that it was possible to prepare a fermented IMP broth with an IMP concentration of 70 g / L or more (FIG. 3). Example 3: Examination of the cell growth rate in case the fungal fermentation product is used in bacterial fermentation
    [0070] [070] In order to confirm the advantage of the case where the first fermentation phase for fungal fermentation and the second fermentation phase for bacterial fermentation are carried out continuously, the growth rate and the degree of proliferation of the bacterial cells were examined , in the case where the vegetable protein degraded through the fungal fermentation phase is added as a source of nutrients and in the case where the vegetable protein is not added.
    [0071] [071] As a result, as can be seen in FIG. 4, in the case in which the degraded vegetable protein was added by the first fermentation stage as a source of nutrients, the bacterial cells proliferated at a high rate and in large quantities compared to the cells in the case in which the vegetable protein was not added (FIG. 4). Example 4: Examination of the alteration of the characteristic aroma of fermented glutamic acid broth according to the type of carbon source
    [0072] [072] Carbon sources that can be used in the fermentation of glutamic acid include glucose, fructose, cane molasses, raw sugar, etc. Although some other components of the medium vary depending on the type of carbon source, the concentration of glutamic acid in the resulting fermented broth does not depend significantly on the type of carbon source. However, the aroma of the fermented broth is significantly altered according to the type of carbon source, and to develop a kokumi aroma, the resulting fermented broth preferably has a cleaner aroma. Cane molasses is an excellent medium component in terms of microbial fermentation due to the various inorganic ions contained in it, but it has a deficiency in that the use value of this as a kokumi aroma is weak due to the color of the medium itself being too dark and remain an aroma caused by caramelization in the resulting culture broth.
    [0073] [073] Thus, raw sugar was used instead of cane molasses in the preparation of the fermented glutamic acid broth for the preparation of a neutral aroma and the resulting change in the characteristic aroma of the culture broth was examined. In this case, while the cane molasses was replaced by raw sugar, inorganic ions and vitamins contained in the cane molasses were additionally added to the medium. As a result, as can be seen, when the cane molasses was replaced by raw sugar, the defective aroma was removed and a characteristic aroma appeared, suitable for the preparation of a kokumi aroma (Table 13). In addition, the fermented glutamic acid broth prepared with raw sugar also contained a high concentration (96 g / L or more) of glutamic acid, which is a concentration of glutamic acid suitable for the preparation of a kokumi flavor. Table 13 Alteration of the characteristics of the aroma of the culture broth in function of the cane molasses cup and raw sugar
    [0074] [074] Using the fermented grain broth prepared in example 1 and the fermented glutamic acid broth prepared in example 2, a kokumi aroma was prepared.
    [0075] [075] Specifically, in order to increase the yield of a filtration process to be performed later, each of the fermented broths was heated to 70 ~ 90 ° C for 15 minutes or more to induce cell lysis. This pre-treatment process can increase the filtration yield to 85% or more. Then, to remove defective aromas and odors from the fermented glutamic acid broth and fermented IMP broth, activated carbon was added in an amount of 1-3% (w / v) based on the volume of the fermented broth, which was then treated with activated carbon at 50 to 70 ° C for 224 hours. After treatment with activated carbon, 1-3% (w / v) diatomaceous broth was added to the fermented broth as a filtration aid, and then filtered through a filter press. Then the fermented broth was used, fermented by the described process.
    [0076] [076] However, when a fungal plant protein source is fermented, the content of low molecular weight peptides and amino acids in the protein source increases. In other words, the content of low molecular weight peptides, with a molecular weight equal to 2000 Da or less, is 30% or more, and preferably, the content of low molecular weight peptides, with a molecular weight of 1500 DA or less, is 40-60%. In addition, the protein source has low molecular weight peptides, each consisting of an average of 14 or less amino acid residues. When fermented grain broth, produced by fungal fermentation, is used as a source of nutrients in bacterial fermentation and is finally properly integrated, a kokumi aroma consisting of nucleic acids can be produced: glutamic acid: peptides in a proportion of about 1: 1: 1 to 1: 1: 5. Specifically, fermented grain broth, IMP fermented broth and glutamic acid fermented broth were mixed and reacted at 70 ~ 100 ° C for 0.5-24 h. Once the reaction was complete, refined salt and dextrin were added to the reaction solution until it reached a 35-50% solids content and the mixture was spray dried and vacuumed to form a powder or concentrated to a solids content of 35-50%. 70% to form a paste. The kokumi aroma can improve the physical feel of a product and can reveal the effect of enhancing the salty taste. Example 6: Evaluation of the sensory attributes of the application of the kokumi aroma
    [0077] [077] When the kokumi aroma of the present invention is applied, it can improve the physical sensation of a product, which designates the texture and weight sensation felt in the mouth, and can reveal the effect of enhancing the salty flavor of the product. In order to verify these effects, the sensory attributes were evaluated. 6-1: Low salt soybean paste
    [0078] [078] In the case of a low salt salt fermented soybean paste, the delicate aroma is weak and particularly the intermediate physical sensation becomes weak while the balance of flavors breaks down and, for this reason, the overall flavor is weak. To confirm the effects of the kokumi aroma on improving the physical sensation and salty taste of the soybean paste, a conventional soybean paste, a low salt soybean paste and a soybean paste comprising 0.3% (p / p) of the kokumi aroma and the panels carried out a sensory evaluation of the global flavor of the soybean pastes,
    [0079] [079] Sensory attributes, such as preference and intensity, were assessed on a 5-point scale. As a result, as shown in Table 17 below, the low-salt soybean paste showed a decrease in overall sensory attributes compared to conventional soybean paste, but the soybean paste comprising kokumi aroma revealed an increase in sensory attributes. . Specifically, the delicate aroma is an attribute that influences the intermediate physical sensation of the food and constitutes an essential attribute of the soybean paste. The intensity and preference of the delicate aroma were weak in the case of low-salt soybean paste, but higher in the case of soybean paste added with kokumi. In addition, in the case of soybean paste added with kokumi, the intensity and preference of the salty taste were higher than in the case of conventional soybean paste, suggesting that the kokumi flavor may increase the body sensation and salty taste of the soybean paste.
    [0080] [080] To confirm the effect of the kokumi aroma to improve the salty flavor, mushroom cream soup comprising 0.1% (pp / p) of added kokumi flavor was presented to the panels, and the panels assessed the perception of intensity of salty flavor in mushroom cream soup. The panels consisted of professional panels trained to recognize the salty taste at all. The sample was supplied at a temperature between 40 and 50 ° C and the sample was presented in triplicate. The mean value of the perception of the salty flavor intensities was analyzed by ANOVA. When assessing the perception of salty flavor intensity, a reference scale was applied in order to eliminate subjective variations.
    [0081] [081] As a result, as can be seen in FIG. 18, in the case of soup with added kokumi / KF aroma, the perception of salty flavor intensity increased by 14.5% compared to soup without added kokumi aroma. Despite the fact that the kokumi aroma, prepared by the salt-free process, did not influence the salt content of the product, it is possible to observe that the kokumi aroma had the effect of increasing the salty flavor of the product.
    权利要求:
    Claims (20)
    [0001]
    Method for preparing a natural kokumi aroma, characterized by comprising: (a) fermenting a vegetable protein source with Aspergillus soybean to obtain a fermented grain broth; (b) fermentation of the fermented grain broth with the microorganisms Corynebacterium sp. or Brevibacterium sp. to prepare a fermented broth of glutamic acid and a fermented broth of inosine-5'-monophosphate (IMP); and (c) mixing the fermented grain broth from phase (a) and fermented broth from phase glutamic acid (b), reacting at a temperature between 70 and 100 ° C for 0.5 to 24 hours.
    [0002]
    Method according to claim 1, characterized in that the natural flavor is prepared using only the fermented broth.
    [0003]
    Method according to claim 1, characterized in that the natural flavor is prepared using the fermented broth without subjecting the fermented broth to an additional chemical process.
    [0004]
    Method according to claim 1 or 2, characterized in that the source of vegetable protein is selected from the group consisting of soy, corn, rice, wheat and wheat gluten.
    [0005]
    Method according to claim 1, characterized in that the microorganism Corynebacterium sp. be Corynebacterium ammoniagenes.
    [0006]
    Method according to claim 1, characterized in that the microorganism Corynebacterium sp. be Corynebacterium glutamicum.
    [0007]
    Method according to claim 1, characterized in that the fermented grain broth and the fermented glutamic acid broth are mixed in a ratio of 1: 0.1 to 1:10.
    [0008]
    Method according to claim 1, characterized in that a composition of the medium for bacterial fermentation intended for the preparation of the IMP fermented broth or the glutamic acid fermented broth comprises a food grade material.
    [0009]
    Method according to claim 8, characterized in that the composition of the medium for bacterial fermentation comprises calcium pantothenate (CAPA).
    [0010]
    Method according to claim 1, characterized in that a means for bacterial fermentation for the preparation of fermented broth of glutamic acid comprises glucose, fructose, raw sugar, betaine, magnesium sulfate, potassium phosphate and phosphoric acid.
    [0011]
    Method according to claim 1, characterized in that a means for bacterial fermentation for the preparation of fermented IMP broth comprises glucose, fructose, magnesium sulfate, phosphoric acid, potassium hydroxide and fermented grain broth.
    [0012]
    Method according to claim 1, characterized in that a carbon source in a medium for bacterial fermentation, intended for the preparation of the fermented broth of glutamic acid is raw sugar.
    [0013]
    Method according to claim 1, characterized in that the concentration of glutamic acid in the fermented broth of glutamic acid of step (b) is between 75 g / L to 150 g / L.
    [0014]
    Method according to claim 1, characterized in that the content of glutamic acid in solid state in the fermented broth of glutamic acid of phase (b) amounts to 50% by weight or more.
    [0015]
    Method according to claim 1, characterized in that the concentration of IMP in the fermented IMP broth of step (b) is between 50 g / L to 150 g / L.
    [0016]
    Method according to claim 1, characterized in that the IMP content in solid state in the fermented IMP broth of phase (b) makes up 30% by weight or more.
    [0017]
    Method according to claim 1, characterized in that it further comprises the treatment of the fermented broth of IMP and the fermented broth of glutamic acid with activated carbon before step (c).
    [0018]
    Method according to claim 17, characterized in that it further comprises the centrifugation or filtration of the treated fermented broth after treatment of the broth with activated carbon.
    [0019]
    Method according to claim 17, characterized in that it further comprises, before treating the fermented broth with activated carbon, heating the fermented broth to a temperature between 70 and 90 ° C for 15 to 60 minutes to induce cell lysis.
    [0020]
    Method according to claim 1, characterized in that it further comprises concentrating the fermented broth and drying the concentrate to prepare powder in step (c).
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    公开号 | 公开日
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    TW201503828A|2015-02-01|
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    JP2015536671A|2015-12-24|
    KR101500847B1|2015-03-16|
    JP2017060469A|2017-03-30|
    AU2014293958A1|2015-12-17|
    SG11201501970QA|2015-04-29|
    AU2014293958B2|2017-05-11|
    EP2900085A4|2016-06-01|
    PH12015500628A1|2015-05-11|
    CN104780780A|2015-07-15|
    TWI556749B|2016-11-11|
    KR20150011700A|2015-02-02|
    WO2015012465A1|2015-01-29|
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    法律状态:
    2018-05-02| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-04-14| B15K| Others concerning applications: alteration of classification|Free format text: A CLASSIFICACAO ANTERIOR ERA: A23L 1/23 Ipc: A23J 3/14 (2006.01), A23L 11/00 (2016.01), A23L 11 |
    2020-04-22| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-07-28| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-12-01| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 25/02/2014, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    KR10-2013-0086971|2013-07-23|
    KR1020130086971A|KR101500847B1|2013-07-23|2013-07-23|Method for preparing natural kokumi flavor|
    PCT/KR2014/001489|WO2015012465A1|2013-07-23|2014-02-25|Method for preparing natural kokumi flavor|
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