Plant-based protein substitute food obtained through immersion culturing of morchella esculenta mycelium, and method for producing same

Abstract

The present invention relates to a plant-based protein substitute food produced through immersion culturing of Morchella esculenta mycelium, and a method for producing same. The mycelium of Morchella esculenta Morel KK (accession No: KACC83080BP) newly isolated for a protein substitute obtained by the method according to the present invention has a short culturing period of 5.5 days compared to other mushrooms, has a remarkably high mycelial production yield 3-4 times greater than that of other mushrooms, has a texture similar to that of chicken or beef after being cooked as a press-molded product, and is remarkably price competitive compared to commercially available plant-based artificial meat, and thus can be effectively used in the development of protein substitute and meat substitute foods.

Description

Plant-based alternative protein food and its production method using immersion culture of Gombo mushroom mycelia

[0001] The present invention relates to a plant-based alternative protein food and a method for producing the same through submerged culture of the mycelia of a newly isolated Morchella esculentaMorel kk; Accession No.: KACC83080BP.

[0002] Alternative proteins are alternative foods intended to replace traditional meat, and are categorized into cultured meat made from animal muscle stem cells, insect protein, seaweed protein, and alternative meat made from proteins extracted from soybeans, wheat, pumpkin, mushrooms, etc.

[0003] Cultured meat refers to producing meat from animal cells using biological technology, and meat substitute refers to a product that can replace meat and imitates the taste and texture of meat using plant-based proteins, etc.

[0004] Meat substitutes are artificially manufactured proteins through plant or grain extraction or microbial cultivation to replicate the taste and texture of animal protein, and are popular among consumers with diverse eating habits, including vegetarians and vegans.

[0005] The alternative protein market has already entered a period of growth not only in the US and Europe, but also in nearby China and Japan. Companies like Impossible Foods, EatJust, and Memphis Meats in the US, Aleph Farms and Future Meat in Israel, and Mosamid and Metble in the Netherlands are leading the charge, establishing themselves as a massive industry. The alternative protein food industry began in 2009 with Beyond Meat's plant-based hamburger patty. Furthermore, in 2013, Mosamid in the Netherlands introduced a cultured meat burger. EatJust, the US company that first successfully commercialized cultured meat technology, developed the world's first eggs made from mung beans in 2017, selling over 100 million eggs in just three years. This achievement has led to its current corporate value of $1.2 billion. Memphis Meat, a cultured meat startup, also made headlines by attracting $17 billion in investment from Bill Gates.

[0006] As of 2021, the alternative protein market has grown to approximately USD 16.7 billion globally and is expected to grow at an average annual rate of 15.3% from 2021 to 2028, with the overall alternative protein market size projected to grow to USD 44.8 billion by 2028.

[0007] On the other hand, in Korea, where alternative proteins are now in the introduction stage, there is an urgent need for deregulation and institutional reform to create a sustainable ecosystem.

[0008] Mushroom mycelium is a low-calorie food source of alternative protein, composed of water, protein, and fiber. When cultured submerged in liquid media, mushroom mycelium offers the advantage of rapid, reliable mass production using automated equipment.

[0009] However, not all mushroom mycelia can be utilized as a meat substitute. These mycelia require a short cultivation period, high mycelial yields, and the resulting mycelia must be able to produce a meat-like texture when cooked. Therefore, the discovery of mushroom mycelia varieties suitable for use as a meat substitute is urgently needed.

[0010] In order to solve the above problems, the present invention provides a method for submerged cultivation of a newly isolated mycelium of Morchella esculentaMorel kk; Accession No.: KACC83080BP) for producing a plant-based alternative protein food composition.

[0011] In addition, the present invention aims to provide a method for processing a newly separated submerged cultured gombo mushroom mycelia to make it suitable as an alternative protein or alternative meat food material.

[0012] In addition, the present invention sought to provide a plant-based alternative protein or alternative meat food processed by the above method.

[0013] One aspect of the present invention relates to a method for producing a meat substitute composition, comprising the steps of: a) culturing newly isolated mycelia of Morchella esculenta Morel kk (Accession No. KACC83080BP) in a medium containing a carbon source, a nitrogen source, and minerals; and b) isolating the mycelia of the Morchella esculenta after culturing.

[0014] Another aspect of the present invention relates to a meat substitute manufactured by the above manufacturing method.

[0015] Another aspect of the present invention relates to a meat substitute food comprising the above meat substitute as an active ingredient.

[0016] The mycelia of the newly isolated morel mushroom (Morchella esculenta) Morel kk (Accession Number: KACC83080BP) obtained for the alternative protein through the present invention are significantly 3 to 4 times more productive than those of other mushrooms, and the texture of the pressed molded product after cooking is similar to that of chicken or beef, and the price competitiveness is significantly superior to that of commercially available plant-based artificial meat, so that they can be usefully utilized in the development of alternative proteins and alternative meat foods.

[0017] Figure 1 is a pressed molded product of the mycelia of the submerged cultured Morchella esculenta Morel kk (Accession number: KACC83080BP).

[0018] FIG. 2 relates to a protein supplement and a meat substitute cooked product processed with a pressed mycelia of submerged cultured Morchella esculenta Morel kk (Accession No.: KACC83080BP).

[0019] The present invention will be described in detail with reference to the attached tables and drawings below.

[0020] If drawings are included, they are provided as examples to ensure that the spirit of the present invention is fully conveyed to those skilled in the art. Therefore, the present invention is not limited to the drawings presented and may be embodied in other forms, and the drawings may be exaggerated to clarify the spirit of the present invention.

[0021] In this case, if there is no other definition in the technical and scientific terms used, they have the meaning commonly understood by a person of ordinary skill in the technical field to which this invention belongs, and the description of known functions and configurations that may unnecessarily obscure the gist of the present invention in the following description and attached drawings are omitted.

[0022] Additionally, the singular forms used in the specification of the present invention may be intended to include the plural forms as well, unless the context specifically indicates otherwise.

[0023] In addition, units used in the specification of the present invention without special mention are based on weight, and for example, units of % or ratio mean weight% or weight ratio.

[0024] Furthermore, in the specification of the present invention, the expression “comprises” is an open description equivalent to expressions such as “includes,” “contains,” “has,” or “is characterized by,” and does not exclude additional unrecited elements, materials, or processes. Furthermore, the expression “consists substantially of…” means that other unrecited elements, materials, or processes may be present together with the specified elements, materials, or processes in an amount that does not significantly affect at least one basic and novel technical idea of ​​the invention to an unacceptable degree. Furthermore, the expression “consists of” means that only the recited elements, materials, or processes are present.

[0025] In the present invention, “sample” or “specimen” refers to an object for analysis and is used with the same meaning throughout the specification.

[0026] Hereinafter, a method for manufacturing the substitute meat composition of the present invention will be described in detail.

[0027] One aspect of the present invention relates to a method for producing a meat substitute composition, comprising the steps of: a) culturing the mycelia of a newly isolated Morchella esculenta Morel kk (Accession No.: KACC83080BP) in a medium containing a carbon source, a nitrogen source, and minerals; and b) isolating the mycelia of the Morchella esculenta Morel kk after culturing.

[0028] The type of mushroom is not particularly limited as long as it can produce an alternative protein or alternative meat of the present invention that is very similar to the texture and texture of actual meat, but is preferably a morel mushroom, more preferably a morel mushroom isolated from the soil of a mountain in Korea, and most preferably a mountain in Gangwon-do, Korea. When the isolated morel mushroom is utilized, the cultivation time is not long compared to when other mushrooms are used, and 3 to 4 times more mycelia can be obtained compared to other types of mushrooms or morel mushrooms isolated from other regions, and is also very similar to the texture and texture of actual meat, so it is preferable.

[0029] The morel mushroom (morel) is an edible mushroom belonging to the morel family of the morel order of the Ascomycetes order. Its scientific name is Morchella. In English-speaking countries, it is commonly called morel, and in Korea, it is called "morel mushroom" due to the resemblance of its surface to a morel (National Institute of Biological Resources, http: / species.nibr.go.kr).

[0030] Morels grow in clusters under trees in forests or gardens from March to May, and the cap of their fruiting bodies contains ascospores and lateral filaments. The body of the morels consists of a cap and a stipe, and is approximately 6–12 cm tall. The cap is pale yellow, broadly ovate, has basket-shaped grooves, and has soft flesh. The stipe is 4–5.5 cm long and 2.6–3 cm wide, nearly cylindrical, white or pale yellowish white, and hollow inside.

[0031] Morel mushrooms are widely distributed in temperate regions of the Northern Hemisphere, including Korea, and are a popular high-quality food ingredient in Europe and North America due to their excellent texture and nutritional value. They have been studied in terms of food and nutritional aspects or artificial cultivation technology, but the application of morel mushroom mycelia cultured under submersion using the newly isolated morel mushroom (Morchella esculenta) Morel kk (Accession Number: KACC83080BP) as an alternative protein material was attempted for the first time in the present invention.

[0032] The above-mentioned Morchella esculenta Morel kk (Accession No.: KACC83080BP) was collected from a mountain in Daehwa-ri, Daehwa-myeon, Pyeongchang-gun, Gangwon-do, isolated after tissue culture, and identified through genetic analysis (Macrogen, Korea). It was named Morchella esculenta Morel kk and deposited with the National Institute of Agricultural Sciences (KACC) on July 3, 2023, with Accession No. KACC83080BP.

[0033] The type or properties of the medium for culturing the newly isolated Morchella esculenta Morel kk (Accession No.: KACC83080BP) are not particularly limited, but it is preferable to use a medium suitable for the combination of the nutrients described below and the cultivation of the Morchella esculenta mycelia, and according to one specific example of the present invention, it may be a liquid medium, and as a more specific example, an immersion culture method may be used in which the nutrients described below are mixed in a solvent such as water at a certain ratio relative to the weight of the entire composition and the mycelia are then immersed and cultured. When the medium is used for immersion culture, the supply of nutrients and the cultivation of the Morchella esculenta are smooth, so that the mycelia of the Morchella esculenta can grow quickly within a short period of time, which is preferable.

[0034] On the other hand, in the case of the gombo mushrooms isolated from other regions, the growth rate of the mycelia was significantly slower than that of the gombo mushrooms newly isolated in the present invention, indicating a significant difference in metabolic activity.

[0035] Nutrients included in the medium for culturing the above-mentioned Morchella esculenta Morel kk (Accession No.: KACC83080BP) may include, but are not limited to, a carbon source, a nitrogen source, minerals, etc., and among these, the carbon source may be one or two or more selected from the group consisting of maltose, glucose, lactose, starch, sucrose, fructose, and galactose, and according to a specific example according to one embodiment of the present invention, based on the total composition, sucrose and fructose, which are disaccharides with a high mycelial growth rate, may be included in an amount of 0.5 to 2% (w / v), preferably 0.7 to 1.6% (w / v), and more preferably 0.9 to 1.1% (w / v), but are not limited to, and within the above range, The mycelium grows quickly, and the amount of mycelium produced increases by 3 to 4 times compared to other mushrooms or mushrooms isolated from other regions.

[0036] Among the nutrients included in the medium for culturing the above-mentioned Morchella esculenta Morel kk (Accession No.: KACC83080BP), the nitrogen source may be one or two or more selected from the group consisting of yeast extract, soy peptone, soy flour, ammonium chloride, calcium nitrate, potassium nitrate, and sodium nitrate, and according to a specific example according to one embodiment of the present invention, based on the total composition, the yeast extract and soy peptone may each be included in an amount of 0.1 to 1% (w / v), preferably 0.15 to 0.5% (w / v), and more preferably 0.20 to 0.30% (w / v), and at the same time, the yeast extract:soy peptone may be present in a ratio of 1:0.6 to 1.5, preferably It may satisfy a ratio of 1:0.8 to 1.3, more preferably a ratio of 1:0.9 to 1.1, but is not limited thereto, and within the above range, the growth of the mycelia of the Gombo mushroom is fast, and the amount of mycelia produced is increased by 3 to 4 times or more compared to Gombo mushrooms isolated from other mushrooms or other regions.

[0037] Among the nutrients included in the medium for culturing the above-mentioned Morchella esculenta Morel kk (Accession No.: KACC83080BP), the minerals may be one or more selected from the group consisting of K2HPO4, KH2PO4, ZnSO4, MgSO4, CuSO4, and FeSO4, but are not limited thereto. According to a specific example according to an embodiment of the present invention, KH2PO4 and MgSO4 used as buffers may be included in an amount of 0.01 to 0.2% (w / v), preferably 0.05 to 0.15% (w / v), and more preferably 0.09 to 0.19% (w / v), based on the total composition, and at the same time, KH2PO4:MgSO4 may satisfy a ratio of 1:0.5 to 2, preferably a ratio of 1:0.7 to 1.5, and more preferably a ratio of 1:0.9 to 1.1, but are not limited thereto. It is not.

[0038] The device or environment for culturing the above-mentioned Morchella esculenta Morel kk (Accession No.: KACC83080BP) is not particularly limited, but may be performed in a stirred bioreactor, and as an operating condition, according to a specific example according to an embodiment of the present invention, for example, it may satisfy the conditions of air supply amount: 0.05 to 0.2 vvm, stirring speed: 30 to 100 rpm, culture temperature: 22 to 27°C, pH 5 to 7, and culture time 4.5 to 7 days, and preferably, it may satisfy the conditions of air supply amount: 0.07 to 0.15 vvm, stirring speed: 45 to 80 rpm, culture temperature: 23 to 26°C, pH 5.4 to 6.6, and culture time 5.0 to 6.2 days, and more preferably, air supply amount: 0.09 to 0.11 vvm, stirring speed: 55 to 65 rpm, culture temperature: 24 to 26℃, pH 5.8 to 6.2, and culture time 5.3 to 5.8 days, but is not limited thereto, and under the above conditions, the growth of the newly isolated Gombo mushroom mycelia is fast, and the amount of mycelia produced is increased by 3 to 4 times or more compared to Gombo mushrooms isolated from other mushrooms or other regions, and it is good that the meat quality and texture similar to actual meat can be obtained. On the other hand, Gombo mushrooms isolated from other mushrooms or other regions have many aspects that feel different from actual meat in terms of meat quality and texture, and as mentioned above, the growth of the mycelia is slow, so they are not good to be used as a substitute meat or substitute meat food.

[0039] Hereinafter, a meat substitute prepared through a method for preparing a meat substitute composition of the present invention and a meat substitute food product containing the same will be described in detail.

[0040] Another aspect of the present invention relates to a meat substitute manufactured by the above manufacturing method.

[0041] The meat substitute manufactured through the above manufacturing method may have a moisture content of 82 to 88%, preferably 83 to 87%, and even more preferably 84 to 86%, but is not limited thereto. When the moisture content is within the above range, the meat quality and texture of the meat substitute are so similar as to be difficult to distinguish from those of actual meat, which is desirable. However, when the moisture content exceeds the above range, the binding force of the meat substitute is reduced, making it difficult to implement the meat quality and texture of meat. When the moisture content falls below the above range, the binding force of the meat substitute is good, but the tissue becomes tough, which is not desirable.

[0042] Another aspect of the present invention relates to a meat substitute food comprising the above meat substitute as an active ingredient.

[0043] The above-mentioned meat substitute food may include, but is not particularly limited to, spices, sweeteners, sauces, and quality improvers. Spices may be used to remove odors (deodorize) when manufacturing plant-based meat substitutes, and specific examples thereof may include, but are not limited to, one or more selected from the group consisting of black pepper (e.g., black pepper powder), garlic (e.g., garlic powder), ginger (e.g., ginger powder), onions (e.g., onion powder), etc. Sweeteners are not particularly limited as long as they provide a refreshing and sweet taste when added and can have the effect of maximizing sweetness when included in manufacturing meat substitutes, and examples thereof include hot water extracts of dried plant fruits such as yacon extracts. In addition, "sauce" in the present specification may generally mean, but is not limited to, a flavored liquid that is marinated to flavor or soften meat before cooking.

[0044] The above-mentioned meat substitute food may be manufactured into various types of formulations selected from the group consisting of steak, katsu, patty, nugget, meatball, sausage, and jerky, but is not limited thereto, and as long as it is in the form of a product that allows the meat quality and texture to be felt as it is, it can be manufactured by cooking without any special restrictions. When manufacturing the above-mentioned meat substitute food, any cooking or manufacturing method that is commonly known in the relevant field (chef, etc.) or a method can be used without limitation, and the cooking method presented in the formulation example of the present invention does not limit the scope of the present invention.

[0045]

[0046] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0047] [Materials and Reagent Information]

[0048] [ingredient]

[0049] - Morel kk (Accession number: KACC83080BP) spawn: Collected from a mountain in Daehwa-ri, Daehwa-myeon, Pyeongchang-gun, Gangwon-do in April 2022, isolated after tissue culture, and used after identification through genetic analysis (Macrogen, Korea).

[0050] [reagent]

[0051] - Other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used.

[0052]

[0053] [Example 1] Exploration of Submerged Culture Conditions for Gombo Mushroom Mycelia

[0054] In the present invention, submerged culture conditions were explored using a 50L bioreactor installed at the Korea Bio-Industry Promotion Agency (Jinju, Republic of Korea).

[0055] 40 L of tap water was added to a 50 L bioreactor, and carbon sources, nitrogen sources, and minerals were added in combination for each experimental group. Then, the mixture was autoclaved for 15 minutes at 121°C and 1.25 bar, cooled to 25°C, and inoculated into a 1,000 mL Erlenmeyer flask with 400 mL of 1% (w / v) newly isolated Morchella esculenta Morel kk (Accession Number: KACC83080BP) spawn.

[0056] 1-1. Measuring the impact of carbon sources

[0057] The effects of the following six carbon sources on the growth of the mycelia of the Gombo mushroom were measured.

[0058] As carbon sources, 2% (w / v) of maltose, glucose, lactose, starch, sucrose, and fructose were added to each experimental group.

[0059] A small amount of 0.1% (w / v) yeast extract was added as a common nitrogen source to each experimental group.

[0060] 10 mL of the liquid spawn of the Gombo mushroom was inoculated into 600 mL of culture water and cultured at 25°C and 120 rpm for 5 days. Afterwards, 100 mL of the culture solution was collected and centrifuged at 10,000 rpm for 10 minutes, the supernatant was removed, and the weight of the mycelia was measured at a moisture content of 85%. The results are shown in Table 1 below.

[0061] Carbon source Maltose Glucose Lactose Starch Hydroxychloropyridine Mycelia Weight (g / 100ml) 1.36g 2.43g 1.23g 2.48g 2.03g 1.95g

[0062] From the results in Table 1 above, it was confirmed that mycelial production was excellent in the order of starch, glucose, sucrose, and fructose as carbon sources.

[0063] Among these, when disaccharides sucrose and fructose were mixed, excluding starch, a polysaccharide, and glucose, a monosaccharide, the amount of mycelia produced according to the mixing ratio was measured using the same method as above and is shown in Table 2 below.

[0064] Carbon source: Fructose (% w / v)Combination ratio 0.5:1.5 1.0:1.0 1.5:0.5 1.5:1.5 2.0:2.0Mycelia weight (g / 100ml) 2.02g 2.53g 2.23g 2.11g 2.10g

[0065] As a result, when disaccharides such as sucrose or fructose were added in amounts of 2% (w / v) or more, the production of mycelia decreased for an unknown reason, and when the carbon source combinations were added in a ratio close to 1:1 at a concentration of 1% (w / v) each, the mycelia weight was surprisingly the highest compared to when they were used alone.

[0066]

[0067] 1-2. Measurement of the impact of nitrogen sources

[0068] The effects of the following six nitrogen sources most commonly used in microbial culture on the growth of the newly isolated mycelia of Morchella esculenta Morel kk (Accession No.: KACC83080BP) were measured.

[0069] As nitrogen sources, soybean powder, soy peptone, yeast extract, ammonium chloride, calcium nitrate, and sodium nitrate were each added to the liquid medium at 0.4% (w / v), which is 1 / 5 of the carbon source.

[0070] A mixture of 1% (w / v) each of sucrose and fructose was added as a common carbon source to each experimental group.

[0071] 10 mL of the liquid spawn of the Gombo mushroom was inoculated into 600 mL of culture water and cultured at 25°C and 120 rpm for 5 days. Afterwards, 100 mL of the culture solution was collected and centrifuged at 10,000 rpm for 10 minutes, the supernatant was removed, and the weight of the mycelia was measured at a moisture content of 85%. The results are shown in Table 3 below.

[0072] Nitrogen source composition 0.4% (w / v) soybean powder soy peptone yeast extract ammonium chloride calcium nitrate sodium nitrate mycelia weight (g / 100ml) 2.21g 2.46g 2.37g 2.12g 1.14g 1.18g

[0073] From the results in Table 3 above, it was confirmed that mycelial production was excellent in the order of using soy peptone, yeast extract, soybean powder, and ammonium chloride as nitrogen sources.

[0074] Among these, when soy peptone and yeast extract were mixed, the amount of mycelia produced according to the mixing ratio was measured using the same method as above and is shown in Table 4 below.

[0075] Nitrogen element soy peptone: Yeast extract (% w / v) combination ratio 0.2:0.2 0.1:0.3 0.3:0.1 0.25:0.25 0.30:0.30 Mycelia weight (g / 100ml) 2.49g 2.39g 2.40g 2.43g 2.43g

[0076] As a result, when soy peptone or yeast extract was added at a concentration of 0.2% (w / v) each in a ratio close to 1:1, the surprising result was that the mycelial weight was the highest compared to when either was used alone, although the reason is unknown.

[0077]

[0078] 1-3. Measuring the influence of minerals

[0079] The effects of the following six minerals, most commonly used in microbial culture, on the growth of the newly isolated mycelia of Morchella esculenta Morel kk (Accession No.: KACC83080BP) were measured.

[0080] As for minerals, KH2PO4 and MgSO4, which are used as buffers based on the total amount of culture solution, were added at 0.1% (w / v) each, and CaCl2, FeSO4, ZnSO4, and CuSO4 were additionally added at 0.1% (w / v) each for each experimental group.

[0081] A mixture containing 1% (w / v) of sucrose and fructose was added as a common carbon source to each experimental group, and a mixture containing 0.2% (w / v) of soy peptone and yeast extract was added as a common nitrogen source.

[0082] 10 mL of the liquid spawn of the Gombo mushroom was inoculated into 600 mL of culture water and cultured at 25°C and 120 rpm for 5 days. Afterwards, 100 mL of the culture solution was collected and centrifuged at 10,000 rpm for 10 minutes, the supernatant was removed, and the weight of the mycelia was measured at a moisture content of 85%. The results are shown in Table 5 below.

[0083] Mineral composition (buffer) KH2PO4 0.1% MgSO4 0.1% (buffer) + CaCl2 0.1% (buffer) + FeSO4 0.1% (buffer) + ZnSO4 0.1% (buffer) + CuSO4 0.1% Mycelia weight (g / 100ml) 2.55g 2.56g 2.56g 2.55g 2.56g

[0084] As a result, it was confirmed that there was no significant difference in the amount of mycelia produced according to the type and content of minerals.

[0085]

[0086] [Example 2] Exploration of operating conditions for submerged cultivation of Gombo mushroom mycelia.

[0087] In order to establish operating conditions for the submerged culture of the newly isolated Morchella esculenta Morel kk (Accession Number: KACC83080BP) mycelia, 40 L of culture water was added to a 50 L bioreactor, and a mixture containing 1% (w / v) each of sucrose and fructose as carbon sources, 0.2% (w / v) each of soy peptone and yeast extract as nitrogen sources, and 0.1% (w / v) each of KH2PO4 and MgSO4 as minerals was added. After inoculating 400 mL of Morchella esculenta spawn, the mixture was cultured for 5 days.

[0088] 2.1 Stirring speed

[0089] In mycelial submersion culture, the agitation speed affects the occurrence of mycelial tangling and the increase in activity. The results of measuring mycelial production after culturing Gombo mushrooms using the same method as in the above example while maintaining different agitation speeds in the incubator are shown in Table 6 below.

[0090] Stirring speed (rpm)Mycelial mass (g / 100ml)401.81502.45602.48702.47802.221001.64

[0091] As a result, it was confirmed that the amount of mycelia produced was high when stirring at 50 to 70 rpm. In the following experiments, stirring was performed at 60 rpm.

[0092]

[0093] 2.2 Air supply

[0094] In order to measure the effect of air supply on mycelial growth in mycelial submersion culture, the amount of mycelial production after culture was measured while maintaining different air supply amounts under the conditions of 2.1 above, and the results are shown in Table 7 below.

[0095] Air supply (vvm) Mycelial mass (g / 100ml) 0.05 2.43 0.10 2.51 0.15 2.50 0.20 2.47

[0096] As a result, the amount of mycelia produced was the highest when the air supply was 0.10 vvm, and when the air supply was 0.15 vvm or more, the generation of bubbles increased as the air supply increased, which caused the injection of antifoaming agent, and accordingly, the amount of mycelia produced also decreased. In the following experiments, the supply was 0.10 vvm.

[0097]

[0098] 2.3 Incubation temperature

[0099] In order to measure the effect of culture temperature on mycelial growth in mycelial submersion culture, the amount of mycelial production after culture was measured while maintaining different culture temperatures under the conditions of 2.2 above, and the results are shown in Table 8 below.

[0100] Temperature (℃) Mycelial mass (g / 100ml) 222.31232.44242.51252.53262.52272.34

[0101] As a result, it was confirmed that the highest amount of mycelia was produced at a culture temperature of 24-26℃. In the following experiments, culture was performed at a temperature of 25℃.

[0102]

[0103] 2.4 Culture medium pH

[0104] In order to measure the effect of the pH of the culture medium on the growth of mycelia in mycelia submerged culture, the amount of mycelia produced after culture was measured while maintaining the pH of the culture medium differently under the conditions of 2.3 above, and the results are shown in Table 9 below.

[0105] pH mycelial mass (g / 100ml) 52.23 5.52.50 62.52 6.52.52 72.49

[0106] As a result, it was confirmed that the amount of mycelia produced was the highest when the pH of the culture medium was 6 to 6.5. In the following experiments, culture was performed at pH 6.

[0107]

[0108] 2.5 Incubation time

[0109] In order to measure the effect of the incubation time on the growth of mycelia in mycelia submersion culture, the amount of mycelia produced after incubation was measured while maintaining the incubation time at different levels under the conditions of 2.4 above, and the results are shown in Table 10 below.

[0110] Incubation period Mycelial mass (g / 100ml) 72 hours (3 days) 0.55 96 hours (4 days) 1.26 120 hours (5 days) 2.49 132 hours (5.5 days) 2.55 144 hours (6 days) 2.55 168 hours (7 days) 2.49 192 hours (8 days) 2.27

[0111] As a result, it was confirmed that the amount of mycelia produced increased rapidly from 4 days onwards, reached a maximum value at 5.5 days, and then gradually decreased from 7 days onwards.

[0112]

[0113] [Example 3] Separation and shaping of the mycelia of the Gombo mushroom after cultivation

[0114] In order to process the mycelia of the Morchella esculenta Morel kk (Accession Number: KACC83080BP) cultured through the above example into an alternative protein material and store and sell it, the mycelia were separated from the culture solution and formulated through molding processing using the following method.

[0115] 3.1 Dehydration of culture medium and mycelial shaping processing

[0116] For mycelial molding processing, the culture medium and mycelia were first separated using the following method.

[0117] A dehydrator or filter press was used to separate the culture medium and mycelia.

[0118] 1) Separation and mycelial shaping processing using a dehydrator

[0119] After primary dehydration by putting the culture medium into a dehydrator, the dehydrated culture medium was injected into a compression mold of appropriate specifications and pressed until the moisture content reached 85%, and then molded.

[0120] 2) Separation and mycelial shaping processing using a filter press

[0121] After filtering the culture medium through a filter press, the separated culture medium was discharged, and the mycelia stored on the filter press plate were separated separately. The mycelia stored on the plate were pressed and shaped using a press until the moisture content reached 85%.

[0122]

[0123] 3.2 Storage and Archiving

[0124] The mycelia formed through the above process were vacuum-packed in vinyl containers of appropriate specifications for storage, preservation, and distribution, and then stored frozen at low temperatures.

[0125] When mycelia are stored in a refrigerator, the mycelia multiply through metabolic activity, generate heat in the formed mycelia, and deteriorate, making them unusable.

[0126]

[0127] [Formulation Example 1] Vegetable protein supplement

[0128] The mycelia pressed molded product with a moisture content of 85% obtained through Example 3 was freeze-dried to obtain frozen mycelia with a moisture content of 1.5%, and then powder was obtained by pulverizing with an ultrafine powderer (Migang Co., Ltd., Republic of Korea).

[0129] Analysis of the composition of the powder revealed that it comprised approximately 50% protein, 45% fiber, and approximately 5% other components. Because it contained approximately 50% protein, the product was formulated as a protein supplement without a separate protein extraction process.

[0130] At this time, functionality was increased by adding an appropriate amount of vitamins, etc. as auxiliary ingredients of protein supplements, and products were manufactured in the form of powder, granules, capsules, and tablets using a manufacturing method of a typical formulation used in this industry.

[0131]

[0132] [Formulation Example 2] Meat substitute food

[0133] The mycelial compression molded product (hereinafter referred to as “meat substitute”) with a moisture content of 85% obtained through Example 3 was cooked to produce various types of meat substitute foods.

[0134] The numbers recorded in the manufacturing stages of the meat substitute food below do not necessarily indicate the manufacturing order, but are distinguished to clarify the manufacturing process. If there is no problem in manufacturing the meat substitute food even when the order is reversed, the manufacturing process by changing the order is also included.

[0135] 2.1 Substitute Meat Steak

[0136] 200g of mycelial extruded molded product (meat substitute), 1 tablespoon of olive oil, an appropriate amount of salt, an appropriate amount of pepper, 1 sprig of rosemary, an appropriate amount of starch, and spices were mixed, aged in the refrigerator for more than 12 hours, and then heated on a frying pan to make a meat substitute steak.

[0137]

[0138] 2.2 Substitute Meat Katsu

[0139] The following process was used to make meat substitute cutlet using 200g of meat substitute, 1 cup of flour, 2 eggs, 1 cup of breadcrumbs, 1L of cooking oil, an appropriate amount of salt, an appropriate amount of pepper, and spices.

[0140] 1) Put flour and breadcrumbs in separate bowls.

[0141] 2) Break the eggs and add a little salt, pepper and spices to make egg wash.

[0142] 3) Coat the meat substitute in flour and dip it in the egg mixture.

[0143] 4) Take out the meat substitute soaked in egg water and coat it in breadcrumbs.

[0144] 5) Place the breaded meat substitute in a frying pan with cooking oil and fry over medium heat for about 3-4 minutes. Once one side is golden brown, turn it over and fry the other side in the same way.

[0145] 6) Place the fried meat substitute on a paper towel to remove oil, then pour the katsu sauce over it to complete the meat substitute katsu.

[0146]

[0147] 2.3 Alternative Meat Hamburger Patty

[0148] A meat substitute hamburger patty was made using the following process: 500g of meat substitute, 1 / 2 cup of breadcrumbs, 1 / 2 cup of soy milk, 1 chopped onion, 2 tablespoons of cooking oil, 2 cloves of minced garlic, an appropriate amount of salt, an appropriate amount of pepper, an appropriate amount of starch, and spices.

[0149] 1) In a large bowl, combine the meat substitute, breadcrumbs, soy milk, onion, cooking oil, garlic, salt, pepper, and spices and mix well to form a dough.

[0150] 2) Divide the dough into 10 pieces of 50g each and shape them using a patty mold.

[0151] 3) Place the formed patty in the refrigerator and let it mature for 30 minutes.

[0152] 4) Coat the pan with cooking oil and heat it until hot, then add the aged patty chunks and cook.

[0153] 5) Once one side is cooked, flip the patty over and cook to complete the meat substitute hamburger patty.

[0154]

[0155] 2.4 Alternative Meat Nuggets

[0156] The following process was used to make meat substitute nuggets using 500g of meat substitute, 50g of vegetable margarine, 1 / 4 cup of soy milk, 1 / 2 cup of flour, 1 / 2 teaspoon of salt, an appropriate amount of pepper, an appropriate amount of cooking oil, an appropriate amount of starch, and flavoring.

[0157] 1) Divide the meat substitute into bite-sized pieces weighing approximately 20g each.

[0158] 2) Place margarine, soy milk, salt, pepper, and spices in a bowl and mix well.

[0159] 3) Coat the divided pieces of meat with flour,

[0160] 4) After coating the flour with the seasoning mixture,

[0161] 5) Coat the meat substitute pieces with the mixed seasoning and breadcrumbs.

[0162] 6) Heat the pan with cooking oil and turn up the heat until hot. Add the breaded meat pieces and cook for about 3-4 minutes until evenly cooked through to complete the meat nuggets.

[0163]

[0164] 2.5 Meat substitute dumplings

[0165] Meat substitute balls were made using 150g of meat substitute, 1 / 2 green onion, 1 / 2 teaspoon of minced ginger, 1 teaspoon of minced garlic, 1 tablespoon of soy sauce, 1 / 2 teaspoon of sugar, 1 teaspoon of sesame oil, an appropriate amount of pepper, an appropriate amount of cooking oil, an appropriate amount of starch, and spices, using the following process.

[0166] 1) Mash the substitute meat to the level of ground meat.

[0167] 2) Add finely chopped green onions, ginger, garlic, soy sauce, sugar, sesame oil, pepper, starch and spices to the ground meat substitute and mix well.

[0168] 3) Divide the mixed meat substitute into bite-sized pieces and shape them into round balls.

[0169] 4) Place the meatballs in an oil pot at 180℃ and cook for about 5 minutes until the center is cooked through.

[0170]

[0171] 2.6 Alternative Meat Sausage

[0172] A meat substitute sausage was made using the following process: 500g of meat substitute, 50g of vegetable margarine, 1 onion, 2 cloves of garlic, 1 teaspoon of salt, an appropriate amount of pepper, an appropriate amount of parsley, an appropriate amount of starch, 1 screw sausage skin, an appropriate amount of cooking oil, and spices.

[0173] 1) Mash the substitute meat to the level of ground meat.

[0174] 2) Put the mashed meat substitute, margarine, onion, garlic, salt, pepper, spices, and parsley in a bowl and mix well to make a dough.

[0175] 3) Let the dough rest in the refrigerator for about 30 minutes.

[0176] 4) Push the matured dough into the screw sausage casing so that it does not burst.

[0177] 5) Tie the ends of the sausage skin tightly to prevent the sausage from flowing out.

[0178] 6) The sealed sausage skin was placed in a steamer and steamed at 110℃ for about 20 minutes to make a meat substitute sausage.

[0179]

[0180] 2.7 Meat substitute jerky

[0181] The following process was used to make meat substitute jerky using 500g of meat substitute, 1 / 2 cup of soy sauce, 1 / 4 cup of sugar, 2 tablespoons of minced garlic, an appropriate amount of pepper, an appropriate amount of starch, an appropriate amount of sesame seeds, and spices.

[0182] 1) Finely chop the meat,

[0183] 2) After mixing the seasonings evenly to make the broth,

[0184] 3) Mix the minced meat and broth and leave in the refrigerator for about an hour.

[0185] 4) The aged meat substitute was placed in a mold and pressed to form a 5 to 10 mm thick product.

[0186] 5) After baking the shaped meat substitute in the oven for about 30 minutes,

[0187] 6) The meat substitute jerky was made by drying it in a cool place with the wind blowing for about 24 hours.

[0188]

[0189] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0190] [Accession number]

[0191] Name of depositor: Korea Academy of Agricultural Sciences (KACC)

[0192] Accession number: KACC83080BP

[0193] Date of acceptance: 20230703

[0194]

Claims

1. a) A step of culturing the newly isolated mycelia of Morchella esculenta Morel kk (Accession No.: KACC83080BP) in a medium containing a carbon source, a nitrogen source, and minerals; and b) A step of separating the mycelia of the Gombo mushroom after culturing; A method for producing a substitute meat composition comprising:

2. In paragraph 1, A method for producing a meat substitute composition, wherein the above-mentioned gombo mushroom (Morchella esculenta) Morel kk (Accession number: KACC83080BP) is isolated from soil in a mountain in Gangwon-do, South Korea.

3. In paragraph 1, A method for producing a substitute meat composition, wherein the above culturing is performed by immersion culturing in a liquid medium.

4. In paragraph 1, A method for producing a meat substitute composition, wherein the carbon source is one or more selected from the group consisting of maltose, glucose, lactose, starch, sucrose, fructose, and galactose.

5. In paragraph 1, A method for producing a meat substitute composition, wherein the nitrogen source is one or more selected from the group consisting of yeast extract, soy peptone, soy flour, ammonium chloride, calcium nitrate, potassium nitrate, and sodium nitrate.

6. In paragraph 1, A method for producing a meat substitute composition, wherein the above mineral is one or more selected from the group consisting of K2HPO4, KH2PO4, ZnSO4, MgSO4, CuSO4, and FeSO4.

7. In paragraph 4, A method for producing a meat substitute composition, wherein the carbon source comprises 0.5 to 2% (w / v) of sucrose and 0.5 to 2% (w / v) of fructose based on the total composition, and satisfies a ratio of sucrose:fructose = 1:0.8 to 1.

2.

8. In paragraph 5, A method for producing a meat substitute composition, wherein the nitrogen source comprises 0.1 to 1% (w / v) of yeast extract and 0.1 to 1% (w / v) of soy peptone based on the total composition, and satisfies a ratio of yeast extract:soy peptone = 1:0.6 to 1.

5.

9. In paragraph 6, A method for producing a meat substitute composition, wherein the mineral comprises KH2PO4 0.01 to 0.2% (w / v) and MgSO4 0.01 to 0.2% (w / v) based on the total composition, and satisfies the ratio of KH2PO4:MgSO4=1:0.5 to 2.

10. In paragraph 1, A method for producing a meat substitute composition, wherein the above culturing is performed in a stirred bioreactor.

11. In paragraph 10, A method for producing a meat substitute composition, wherein the operation of the above-mentioned stirred bioreactor satisfies the following conditions. Air supply: 0.05 to 0.2 vvm, stirring speed: 30 to 100 rpm, culture temperature: 22 to 27°C, pH 5 to 7, and culture time: 4.5 to 7 days.

12. Substitute meat manufactured by any one of the manufacturing methods of Articles 1 to 11.

13. In paragraph 12, The above substitute meat is a substitute meat having a moisture content of 82 to 88%.

14. A meat substitute food containing the meat substitute of Article 12 as an effective ingredient.

15. In paragraph 14, The above meat substitute food is a meat substitute food containing spices, sweeteners, sauces and quality improvers.

16. In paragraph 14, A meat substitute food, wherein the form of the above meat substitute food is selected from the group consisting of steak, katsu, patty, nugget, meatball, sausage and jerky.

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