Neurospora intermedia and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to Neurospora intermedia and an application of the Neurospora intermedia. The neurospora intermedia provided by the invention is any one of the following A1)-A4): A1) a strain of which the ITS sequence has at least 98% identity with the sequence as shown in SEQ ID NO: 3; (A2), Neurospora intermedia MN134912, and the preservation number of the Neurospora intermedia MN134912 is GDMCC No.65441; a3) a strain having at least 98% identity with the ITS sequence of the strain in A2); a4), wherein the average nucleotide consistency of the genome of the strain is at least 98% with that of the genome of any one of the strains (A1) to (A3); the hypha protein of the neurospora intermedia has the advantages of high protein, high dietary fiber, low cholesterol and a proper amount of unsaturated fatty acid, is rich in ergothioneine, and can be used for high-yield production of ergothioneine.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Neurospora intermedia and uses thereof. Background Art

[0002] Animal meat is rich in high-quality protein, essential vitamins and minerals, and other nutrients, and plays an important role in the human diet. However, despite the above advantages of animal meat, it also contains high levels of cholesterol and unsaturated fatty acids. Long-term intake of high cholesterol and unsaturated fatty acids may have adverse effects on human health. For example, high cholesterol can easily cause fatty liver, hyperlipidemia, hypertension, atherosclerosis, cerebral infarction and other diseases. Although foods with high levels of unsaturated fatty acids are good for health, excessive intake may increase the risk of cardiovascular disease. Therefore, it is very meaningful to seek a meat substitute protein that contains low cholesterol, moderate amounts of unsaturated fatty acids, and is rich in nutrients needed by humans (such as dietary fiber, trace elements, amino acids, etc.).

[0003] As a new type of protein resource, microbial protein is rich in nutrients, with high protein content in the bacteria, complete amino acid components, especially high essential amino acid content. It is also rich in carbohydrates, vitamins and minerals. Its production is not affected by external conditions such as climate, and its output rate is high. Based on this, the processing of microbial protein into food has become a new research hotspot, among which imitation meat products using microbial protein as raw materials are increasingly attracting the attention of R&D personnel and consumers.

[0004] Neurospora intermedia is a fungus of the genus Neurospora, belonging to the phylum Ascomycoya, class Ascomycetes, order Sordariales, family Sordariaceae. It has been reported in the literature that Neurospora intermedia is used to prepare alternative proteins. Although the biomass obtained by drying its mycelium has a high protein content, the other important nutrients, total dietary fiber and unsaturated fatty acids, are low in content, and the nutrients are not rich, making it difficult to completely replace animal meat. In addition, the cholesterol content has not been studied, and the cholesterol content in mycelial protein has been ignored as an important indicator. Therefore, a mycelial protein of Neurospora intermedia with a low cholesterol content has not yet been developed. The above shows that there is currently no mycelial protein of Neurospora intermedia that simultaneously meets the requirements of high protein, high dietary fiber, low cholesterol, and an appropriate amount of unsaturated fatty acids, which leads to the fact that the existing mycelial protein of Neurospora intermedia may be difficult to meet the nutritional and health needs of humans when it is partially or completely used as a meat substitute protein product.

[0005] Ergothioneine (mercaptohistidine trimethyl salt, ergothioneine, EGT) is a thiol compound with a chemical name of 2-thioimidazole amino acid, and its left-handed structure has good physiological activity. Ergothioneine has two isomeric forms, thiol and thiol, and mainly exists in the form of thiol in aqueous solution. Ergothioneine plays an important role in human cells and tissues, which is mainly reflected in the following aspects: 1. Ergothioneine has excellent antioxidant properties and stability, can remove cell active oxygen, resist oxidative damage and maintain redox balance; 2. Ergothioneine has the potential to prevent and treat diseases such as cancer, cardiovascular disease, cognitive dysfunction caused by oxidative stress; 3. Ergothioneine also has the effect of improving sleep disorders, improving intestinal health, and resisting ultraviolet damage and photoaging. Due to these characteristics of ergothioneine, it is determined that it has a wide range of uses and market prospects in the fields of medicine, food and beverage, functional food, animal feed, cosmetics and biotechnology. Ergothioneine was originally isolated from the ergot fungus of rye in the family Poaceae, and microorganisms such as mycobacteria, methylbacterium, cyanobacteria and mushrooms can synthesize ergothioneine naturally. Ergothioneine has been shown to be present in almost all human cells and tissues, usually in the brain, bone marrow, lens and cornea of ​​the eyes, and red blood cells at millimolar levels, but the human body itself cannot synthesize ergothioneine, and usually can only be taken in and supplemented from food. However, there is no relevant literature that Neurocystis can produce ergothioneine, and it is not disclosed that it can produce ergothioneine in high yield. Summary of the invention

[0006] Therefore, the technical problem to be solved in the present invention is to provide a kind of Neurospora intermedia (Neurospora intermedia) and its use, the mycelial protein of the Neurospora intermedia satisfies the advantages of high protein, high dietary fiber, low cholesterol, and an amount of unsaturated fatty acids at the same time, and is rich in ergothioneine, can be used for high-yield production of ergothioneine, in addition, the fermentation and culture process of the strain can be amplified, and the fermentation time is short, and the commercial production conversion rate is high.

[0007] To this end, the present invention provides the following technical solutions:

[0008] The embodiments disclosed in the present invention relate to a Neurospora intermedia, which is any one of the following A1) to A4):

[0009] A1), a strain whose ITS sequence is at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the sequence shown in SEQ ID NO: 3;

[0010] A2), Neurospora intermedia MN134912, deposited in Guangdong Microbiological Culture Collection Center, with the deposit number of GDMCC No.65441;

[0011] A3) a strain having at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identity with the ITS sequence of the strain described in A2);

[0012] A4), a strain whose genome has an average nucleotide identity of at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% with the genome of any of the strains described in A1) to A3).

[0013] As used herein, "identity" with respect to nucleic acid sequences refers to the extent to which the two nucleic acid sequences have identical nucleotide residues at the same position when the two nucleic acid sequences are aligned to achieve the maximum level of identity, expressed as a percentage. Wherein, the "identity" between the sequences of two nucleic acid molecules can be determined using a known computer algorithm, such as at least one of the GCG program package, BLASTN or FASTA, and other commercial or publicly available programs may also be, for example, DNAStar-"MegAlign" program.

[0014] In addition, with the rapid development of second-generation and third-generation sequencing technologies, strain identification based on whole genome sequencing has become possible, making strain identification results more accurate. Average nucleotide identity (ANI) of bacterial genomes refers to the similarity of homologous genes between two microbial genomes. At present, there are various mature ANI value calculation tools, such as local calculation software Jspecies ( / jspecies) and Gegenees ( / documentation.html), online calculation tools ANI caculator (http: / enveomics.gatech.edu / ), EzGenome ( / ezgenome / ani) and ANItools, etc. Therefore, the ANI value of the isolated strain and MN134912 can be calculated by any of the above local calculation software or online calculation tools or other tools or methods known in the art.

[0015] The culture characteristics of the inter-type Neurospora strain MN134912 are that the colony diameter is 2-6 cm after culturing for 4 days, the colony grows in a spreading manner on the MEA medium, the hyphae are luxuriant and velvety, and the colony is irregular; the conidia are light yellow, the surface of the substrate is white, and the substrate turns orange after being left for a long time; the hyphae morphology is characterized by ascus aggregated or scattered, the ascus is cylindrical, and has a short stalk. The hyphae length is 500-3000 μm, and the hyphae diameter is 1-10 μm. The conidia are orange-yellow, in strings, and spherical to ovoid.

[0016] In some embodiments, the intermorphic Neurospora comprises an ergothioneine synthesis gene; preferably, the intermorphic Neurospora comprises a sequence that is at least 95%, 95.5%, 96.5%, 97%, 97.5%, 98%, 98.29%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the sequence of at least one of the ergothioneine synthesis genes EGT-1 and EGT-2.

[0017] The present invention discloses a microbial agent, including the inter-type Neurospora, its inactivated material, its culture, its fermented material, and / or its extract. The "culture", "fermentation broth", "inactivated material" and "extract" used herein include any components, compounds, substances or by-products (including but not limited to small molecule secondary metabolites) produced by the inter-type Neurospora strain described herein and having any beneficial effect disclosed in the present invention. Any components, compounds, substances or by-products of the beneficial effect include proteins, amino acids, trace elements, dietary fiber, ergothioneine, etc.

[0018] The embodiment of the present invention discloses a mycelial protein or mycelium, which is prepared from the Neurospora intermedia or the microbial agent.

[0019] Preferably, the hyphae length of the hyphae protein or mycelium is 500-3000 μm, and / or the hyphae diameter is 1-10 μm;

[0020] In some embodiments, the hyphal protein or mycelium contains at least one of cholesterol, unsaturated fatty acids, dietary fiber, protein, ergothioneine, amino acids and trace elements.

[0021] In a preferred embodiment, whether measured on a wet basis or a dry basis, the cholesterol content of the mycelial protein or mycelium is <0.9 mg / 100 g, which is much lower than that of chicken (70 mg / 100 g) and beef (84 mg / 100 g), indicating that the mycelial protein or mycelium has a low cholesterol content and is healthier for use in the food field than animal meat such as chicken and beef.

[0022] In some embodiments, the protein content is higher than 45% based on the dry weight of mycelial protein or mycelium. Preferably, the protein content is 50%-65% based on the dry weight of mycelial protein or mycelium, which is much higher than the protein content in chicken and beef (19-21%), indicating that the mycelial protein or mycelium has a high protein content and has a higher nutritional value than animal meat such as chicken and beef.

[0023] In some embodiments, the dietary fiber content is higher than 25% based on the dry weight of mycelial protein or mycelium. Preferably, the dietary fiber content is 30%-40% based on the dry weight of mycelial protein or mycelium, while animal meat such as chicken and beef do not contain total dietary fiber, indicating that the mycelial protein or mycelium has a high total dietary fiber content and has a higher nutritional value than animal meat such as chicken and beef. In some embodiments, the dietary fiber includes at least one of soluble dietary fiber and insoluble dietary fiber.

[0024] In a preferred embodiment, the unsaturated fatty acids include at least one of total monounsaturated fatty acids and total polyunsaturated fatty acids, and the total monounsaturated fatty acid content in the dry basis of the mycelial protein or mycelium is significantly lower than that of animal meat such as chicken and beef. At the same time, the total polyunsaturated fatty acid content in the dry basis of the mycelial protein or mycelium is significantly higher than that of animal meat such as chicken and beef.

[0025] The above description shows that the mycelial protein or mycelium simultaneously meets the requirements of high protein, high dietary fiber, low cholesterol, and appropriate amount of unsaturated fatty acids, is rich in nutrients and has a high content, is healthier, and can be applied in the food field to partially or completely replace animal protein.

[0026] Furthermore, the mycelial protein has excellent filaments, the filter cake is light flesh-colored, tough, and has no obvious odor; therefore, the tasting result is good, indicating that it is very suitable for application in the food field.

[0027] In a preferred embodiment, the hyphal protein also contains ergothioneine, and has the advantage of high yield, based on the dry weight of the hyphal protein or mycelium, the ergothioneine content of the hyphal protein or mycelium is 500-2000 μg / g dry weight, which is significantly higher than the ergothioneine content 466~850 μg / g dry weight in other natural fungi in the genus Neurospora reported, and higher than the dry weight of ergothioneine content 210ng / mg~2600ng / mg (Pleurotus ostreatus) in other fungi. Explanation, the inter-type Neurospora of the present invention can be used for fermentation of high-yield ergothioneine. Further, the hyphal protein or mycelium is applied to the field of food, so that the food is rich in ergothioneine, with the function of ergothioneine, such as anti-oxidation, disease prevention, improvement of sleep and other health functions, and thus, the hyphal protein or mycelium can be used to prepare health food or functional food. Further, according to the multiple functions of ergothioneine, the hyphal protein or mycelium can also be used to prepare cosmetics, medicines, etc.

[0028] The embodiment disclosed in the present invention relates to the use of Neurospora intermedia in the production of mycelial protein or mycelium, and fermentation culture is carried out using the Neurospora intermedia or the microbial agent.

[0029] The embodiment disclosed in the present invention relates to a method for producing mycelial protein or mycelium, comprising fermenting and culturing the Neurospora intermedia or the microbial agent, separating mycelium from the fermentation product, and obtaining mycelial protein.

[0030] In some embodiments, the fermentation culture includes at least one of liquid fermentation and solid fermentation; preferably, it is liquid fermentation.

[0031] In some embodiments, the volume of the liquid fermentation is ≥1 L, and can be any one of 1 L, 5 L, 10 L, 20 L, 30 L, 40 L, 50 L, 100 L, 200 L, 300 L, etc., or a range between any two of the above values. In some embodiments, the inoculum size of Neurospora intermedia is a conventional inoculum size, such as an inoculum size of 1 to 10 v / v% of the total volume of the culture medium.

[0032] In some embodiments, the nitrogen source in the liquid culture medium used in the fermentation culture includes at least one of an inorganic nitrogen source and an organic nitrogen source. The inorganic nitrogen source includes urea and / or ammonium compounds. The organic nitrogen source includes, but is not limited to, a cheap nitrogen source such as corn starch saccharification liquid, and may also be defatted soybean flour, peanut cake powder, corn steep liquor powder, tryptone, etc.

[0033] In some embodiments, the liquid culture medium used in the fermentation culture further includes at least one of a carbon source, trace elements, organic acids, and vitamins.

[0034] In some embodiments, the carbon source includes but is not limited to at least one of sugar, alcohol, honey, and starch.

[0035] Preferably, the carbon source comprises sucrose, maltose, glucose, fructose, glycerol, polyols, corn starch, maltodextrin, cyclodextrin, glucose syrup, modified starch, corn starch saccharification liquid, rice starch saccharification liquid, sweet potato starch saccharification liquid, molasses, hydrogenated glucose syrup, maltitol syrup, sorbitol syrup, beet pulp, agricultural pulp, wood pulp, dried grains, brewery waste, carbon refined syrup, desalted syrup, enzyme-converted syrup and / or a combination thereof; in a preferred embodiment, the carbon source is selected from corn starch saccharification liquid.

[0036] In some embodiments, the trace elements include but are not limited to at least one of potassium, magnesium, calcium, manganese, iron, and zinc; in a preferred embodiment, the potassium source is selected from potassium salts, preferably potassium nitrate and potassium dihydrogen phosphate. The magnesium source is selected from magnesium salts, preferably magnesium sulfate heptahydrate. The calcium source is selected from calcium salts, preferably calcium chloride. The manganese source is selected from manganese salts, preferably manganese sulfate monohydrate. The iron source is selected from iron salts, preferably iron sulfate heptahydrate. The zinc source is selected from zinc salts, preferably zinc chloride. In a preferred embodiment, the K content of the fermentation medium used is 2.5-3.0. + The concentration is 1-10g / L, Mg 2+ The concentration is 0.3-5g / L, Ca 2+ The concentration is 0.01-1g / L, preferably, K + The concentration is 1-5g / L, Mg 2+ The concentration is 0.3-2g / L, Ca 2+ The concentration is 0.01-0.5g / L.

[0037] In some embodiments, the organic acid is selected from citric acid.

[0038] In some embodiments, the vitamins include, but are not limited to, B family vitamins;

[0039] Preferably, the fermentation medium used in the fermentation culture comprises 20 to 35 parts by weight of a carbon source, 0.01 to 30 parts by weight of a nitrogen source and 0.01 to 10 parts by weight of an inorganic salt.

[0040] Preferably, the fermentation medium comprises: 20 to 35 parts by weight of corn starch saccharification liquid; 0.1 to 2 parts by weight of citric acid; 1 to 5 parts by weight of potassium dihydrogen phosphate; 0.01 to 10 parts by weight of ammonium sulfate; 0.3 to 2 parts by weight of magnesium sulfate heptahydrate; 0.01 to 1 parts by weight of calcium chloride; and, optionally, 0.001 to 0.1 parts by weight of trace elements.

[0041] Preferably, the fermentation medium further comprises a vitamin complex.

[0042] In some embodiments, the pH of the liquid culture medium used in the fermentation culture is 4-8, and the pH can be any one of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 or a range between any two of the above values, and the more preferred pH is 6-7. From the above, it can be seen that the pH range is relatively wide, indicating that the Neurospora intermedia has a high pH tolerance, the strain is easy to separate and culture, and is suitable for scale-up culture, fermentation or industrial fermentation, and has a high commercial conversion rate. For another example, strain MN134912 has a stronger alkaliphilicity, and the nitrogen source can be selected from alkaline ammonia water, which is cheaper and saves costs, and can be used as a nitrogen source to supplement the pH value of the mycelium. Under the condition of pH equal to 6-7, the mycelium can grow well and produce high protein.

[0043] In some embodiments, the fermentation culture temperature is 28-40°C, and the temperature can be any one of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C or a range between any two of the above values, and the preferred temperature is 28-32°C. As can be seen from the above, the culture temperature range is relatively wide, indicating that the Neurospora intermedia has a high temperature tolerance, the strain is easy to separate and culture, and is suitable for scaled-up culture, fermentation or industrial fermentation. For example, a high-volume culture or fermentation system is prone to high temperature, and the strain of the present invention has good temperature tolerance, which can ensure that it can still grow and ferment well under high temperature conditions.

[0044] In a preferred embodiment, before fermentation, the process further comprises the steps of activating the Neurospora intermedia, culturing the seeds and / or amplifying the culture.

[0045] In a preferred embodiment, the activation culture is carried out on a solid culture medium. The culture medium used for the activation culture can be any conventional culture medium. In a preferred embodiment, the activation culture medium includes but is not limited to MEA plate culture medium, Sabouraud medium (SDA), and potato dextrose agar medium (PDA). The temperature and time of the activation culture are conventional culture conditions. In some embodiments, the culture temperature is 28-40°C and the culture time is 24-96h, preferably the culture temperature is 28°C and the culture time is 72h.

[0046] In some embodiments, the seed culture medium used in the seed culture comprises at least one of a nitrogen source, a carbon source, a trace element, an organic acid, and a vitamin. The types of the nitrogen source, the carbon source, the trace element, the organic acid, and the vitamin are consistent with the carbon source, the trace element, the organic acid, and the vitamin used in the aforementioned fermentation culture.

[0047] In a preferred embodiment, the seed culture medium comprises: 25 to 35 parts by weight of a carbon source, 10 to 35 parts by weight of a nitrogen source and 0.1 to 2 parts by weight of an inorganic salt.

[0048] In a preferred embodiment, the seed culture medium comprises: 25 to 35 parts by weight of glucose, 10 to 35 parts by weight of yeast extract, 1 to 5 parts by weight of potassium dihydrogen phosphate, 0.3 to 2 parts by weight of magnesium sulfate heptahydrate, and 0.01 to 1 parts by weight of calcium chloride. When preparing the seed culture medium, the priority order of metal ions is K>Mg>Ca. In addition, when the culture medium volume is 50mL to 1L, in a preferred embodiment, KH2PO4 1g / L, MgSO4 0.5g / L, CaCl20.2g / L.

[0049] In a preferred embodiment, the amplified culture is carried out in a liquid culture medium, and the culture medium used can be any conventional culture medium. Further, the culture medium used is a common seed culture medium, and the seed culture medium includes but is not limited to a carbon source, a nitrogen source, and metal ions. Metal ions include but are not limited to potassium ions, magnesium ions, and calcium ions. In a specific embodiment, the culture medium includes but is not limited to glucose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, and calcium chloride. In a more specific embodiment, the seed culture medium contains glucose 25-35g / L, yeast extract 5-35g / L, potassium dihydrogen phosphate 1-10g / L, magnesium sulfate heptahydrate 0.5-5g / L, and calcium chloride 0.2-1g / L. When preparing the seed culture medium, the priority order of metal ions is K>Mg>Ca. In addition, when the culture medium volume is 50mL~1L, in a preferred embodiment, KH2PO4 1g / L, MgSO4 0.5g / L, CaCl2 0.2g / L.

[0050] In a preferred embodiment, the pH of the liquid culture medium for the amplified culture is 4 to 8, and the pH may be any one of 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 or a range between any two of the above values, and the more preferred pH is 6 to 7. As can be seen from the above, the culture temperature range is relatively wide, indicating that the Neurospora intermedia has a high pH tolerance, and the strain is easy to separate and culture, and is suitable for amplified culture, fermentation or industrial fermentation.

[0051] In some embodiments, the conditions for the amplified culture are conventional culture conditions. In some embodiments, the preferred culture temperature is 28-40°C, and the temperature can be any one of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C or a range between any two of the above values, and the preferred temperature is 28-32°C. As can be seen from the above, the culture temperature range is relatively wide, indicating that the Neurospora intermedia has a high temperature tolerance, the strain is easy to separate and culture, and is suitable for amplified culture, fermentation or industrial fermentation.

[0052] In some embodiments, the time of amplified culture can be any time, and the preferred culture time is 24 to 48 hours. In some embodiments, the culture speed is a conventional speed condition, and the preferred speed is 100-300rpm. The amplified culture is at least one level seed liquid culture, and preferably, the amplified culture includes but is not limited to a first level seed liquid culture and a second level seed liquid culture. In some embodiments, the volume of the seed liquid culture can be any volume, and preferably ≥50mL, such as any one of 50mL, 1L, 5L, 10L, 20L, 30L, 40L, 50L, etc., or a range between any two of the above values. In some embodiments, the inoculation amount of the intergenic Neurospora is a conventional inoculation amount, such as an inoculation amount of 1 to 10 v / v% of the total volume of the culture medium.

[0053] In some embodiments, when producing mycelial protein rich in ergothioneine, the culture medium used further includes a nutrient component that promotes the synthesis of ergothioneine.

[0054] In a preferred embodiment, the nutritional components include but are not limited to at least one of histidine and ferrous sulfate;

[0055] In some embodiments, the culture medium used includes a seed culture medium and / or a fermentation culture medium. In a preferred embodiment, in the culture medium used, the concentration of histidine and ferrous sulfate is 0.1-10 g / L. Preferably, the concentration range of histidine and ferrous sulfate is 0.5-2 g / L, and more preferably, the concentration of histidine and ferrous sulfate is 1 g / L.

[0056] An embodiment of the present invention provides a mycelial protein or mycelium obtained by the above method for producing mycelial protein or mycelium.

[0057] The embodiments of the present invention disclose the use of the Neurospora intermedia, the microbial agent, or the hyphal protein or mycelium in producing alternative proteins, alternative protein products, ergothioneine, or products containing ergothioneine.

[0058] In some embodiments, the alternative protein product comprises a food, a condiment, or a feed;

[0059] In some embodiments, the product containing ergothioneine includes food, medicine or cosmetics; optionally, the food includes health products or functional foods;

[0060] In some embodiments, the food includes but is not limited to at least one of a meat analog, a plant-based protein meat product, a dairy product, a beverage, a dietary fiber food, a bakery product, a spreadable product, a health product, or a functional food;

[0061] In some embodiments, the meat analogs include, but are not limited to, meat analogs of livestock, seafood, and poultry;

[0062] In some embodiments, the plant-based protein meat products include, but are not limited to, plant-based protein meat products from livestock, seafood, and poultry;

[0063] In some embodiments, the livestock include but are not limited to at least one of pigs, cattle, and sheep;

[0064] In some embodiments, the seafood includes but is not limited to at least one of fish, shrimps, crabs, and shellfish;

[0065] In some embodiments, the plant-based protein meat products include, but are not limited to, plant-based protein meat products derived from soy protein, pea protein, peanut protein, and wheat protein;

[0066] In some embodiments, the meat analogs and plant-based protein meat products include, but are not limited to, meat sausages, minced meat, meat balls, meat patties, meat-like cuts, and meat-like sliced ​​products such as ham and bacon;

[0067] In some embodiments, the dietary fiber food includes grain product substitutes and fruit and vegetable product substitutes.

[0068] In some embodiments, the health product or functional food includes a protein supplement, an amino acid supplement, or a dietary fiber supplement;

[0069] In some embodiments, the feed comprises poultry feed, livestock feed, aquaculture feed or pet feed.

[0070] The present invention discloses a protein food ingredient comprising a. cultured Neurospora intermedia biomass in an amount of at least 3.8% w / w; and b. water in an amount of up to 96.2% w / w;

[0071] In some embodiments, the Neurospora intermedia is any one of the following A1) to A4):

[0072] A1), a strain whose ITS sequence is at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the sequence shown in SEQ ID NO: 3;

[0073] A2), Neurospora intermedia MN134912, deposited in Guangdong Microbiological Culture Collection Center, with the deposit number of GDMCC No.65441;

[0074] A3) a strain having at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identity with the ITS sequence of the strain described in A2);

[0075] A4), a strain whose genome has an average nucleotide identity of at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% with the genome of any of the strains described in A1) to A3).

[0076] In some embodiments, the Neurospora intermedia biomass is Neurospora intermedia hyphal protein or mycelium;

[0077] In some embodiments, the protein food ingredient comprises 5% w / w-55% w / w of the cultured Neurospora intermedia biomass and 95% w / w-45% w / w water;

[0078] In some embodiments, the protein food ingredient comprises 5% w / w-15% w / w of the cultured Neurospora intermedia biomass and 95% w / w-85% w / w water;

[0079] In some embodiments, the protein food ingredient comprises 90% w / w-99% w / w of the cultured Neurospora intermedia biomass and 1% w / w-10% w / w water;

[0080] In some embodiments, the protein food ingredient further comprises carbohydrates, fat, protein, fiber, or any combination thereof;

[0081] In some embodiments, the carbohydrate comprises glucose, fructose, galactose, maltose, sucrose, lactose, starch, or any combination thereof;

[0082] In some embodiments, the carbohydrate comprises white sugar, brown sugar, black sugar, agar, pectin, gum arabic, molasses, honey, cane sugar, wheat starch, corn starch, potato starch, food starch, dextrose, or any combination thereof;

[0083] In some embodiments, the fat comprises vegetable oil, animal oil, or any combination thereof;

[0084] In some embodiments, the fat comprises safflower oil, sunflower oil, olive oil, coconut oil, sunflower oil, safflower oil, rapeseed oil, canola oil, vegetable glycerin, soy lecithin, or any combination thereof;

[0085] In some embodiments, the protein comprises plant protein, animal protein, microbial protein, or any combination thereof;

[0086] In some embodiments, the protein comprises texturized pea protein, texturized wheat protein, texturized soy protein, soy protein, wheat protein, pea protein, rice protein, potato protein, albumin, or any combination thereof;

[0087] In some embodiments, the fiber includes pea fiber, oat bran, other plant fiber, bamboo fiber, cellulose, citrus fiber, soy fiber, or any combination thereof;

[0088] In some embodiments, the protein food ingredient further comprises soybeans, lentils, lentil flour, soybean flour, pea flour, yeast extract, nutritional yeast (fixed dry yeast), mushroom powder, white button mushroom powder, shiitake mushroom powder, chickpeas, isolated oat products, isolated pea products, fermented rice extract, kelp extract, algae, silicon dioxide, vitamins, dry yeast, citrus extract, beet pulp, beet juice, beet juice extract, turmeric, mushroom extract, shiitake mushroom stems, shiitake mushrooms, white button mushrooms, tofu, soy hydrolyzate, maltodextrin, sodium ascorbate, calcium carbonate, yeast extract, seaweed, spices, sweeteners, natural flavoring agents, or any combination thereof.

[0089] The embodiment of the present invention discloses an edible composition, comprising the hyphal protein or mycelium of the inter-type Neurospora, or the protein food ingredient, and food additives. The amount of the hyphal protein or mycelium or protein food ingredient added is ≥0.01wt% of the total amount. Further, the amount of the hyphal protein or mycelium or protein food ingredient added is 0.05wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 8wt%, 10wt%, 15wt%, 20wt%, 30wt%, 40wt%, 50wt%, 60wt%, 70wt%, 80wt%, 90wt%, 100wt% of the total amount, any one of the values ​​or the range value between any two values. In some embodiments, the food additives, such as plant or animal protein, fat, emulsifier, thickener, stabilizer, flavoring agent, pH adjuster, oil, spices, salt, etc.

[0090] The technical solution of the present invention has the following advantages:

[0091] The present invention provides a Neurospora intermedia, which is any one of the following A1) to A4): A1), a strain whose ITS sequence is at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the sequence shown in SEQ ID NO: 3; A2), Neurospora intermedia MN134912, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No.65441; A3), a strain whose ITS sequence is at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the strain described in A2); A4), a strain whose genome is at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the genome of any one of the strains described in A1) to A3); the hyphal protein or mycelium of the above-mentioned Neurospora intermedia has the advantages of high protein, high dietary fiber, low cholesterol, and appropriate amount of unsaturated fatty acids, is rich in nutrients and has a high content, is healthier, can be used in the food field, and can partially or completely replace animal protein.

[0092] Furthermore, the mycelial protein has excellent filaments, the filter cake is light flesh-colored, tough, and has no obvious odor, so the tasting result is good, indicating that it is very suitable for application in the food field.

[0093] Furthermore, the optimum pH of the Neurospora intermedia is 4-8, and it has a high pH tolerance. The strain is easy to separate and culture, and is suitable for scale-up culture, fermentation or industrial fermentation, and has a high commercial conversion rate. The optimal pH is 6-7, which has stronger alkaliphilicity. The nitrogen source can be selected from alkaline ammonia water, which is cheaper and saves costs. It can also be used as a nitrogen source to supplement the pH value of the mycelium. Under the condition of pH equal to 6-7, the mycelium can grow well and produce high protein.

[0094] Furthermore, the optimum temperature of the Neurospora intermedia is 28-40°C, indicating that the Neurospora intermedia has a high temperature tolerance, and the strain is easy to separate and culture, and is suitable for scale-up culture, fermentation or industrial fermentation.

[0095] Further, the hyphal protein or mycelium of the described inter-type Neurospora also contains ergothioneine, and has the advantage of high yield, and the ergothioneine content in the mycelium reaches 1630.3 μg / g dry weight, which is significantly higher than the ergothioneine content 466~850 μg / g dry weight in other natural fungi in the genus Neurospora reported, and higher than the dry weight of ergothioneine content 210ng / mg~2600ng / mg (Pleurotus ostreatus) in other fungi. Explanation, the inter-type Neurospora of the present invention can be used for fermenting high-yield ergothioneine. Further, the hyphal protein or mycelium are applied to the field of food, so that food is rich in ergothioneine, with the function of ergothioneine, such as anti-oxidation, disease prevention, health care functions such as improving sleep, and thus, the hyphal protein or mycelium can be used for preparing health food or functional food. Further, according to the multiple functions of ergothioneine, the hyphal protein or mycelium can also be used for preparing cosmetics, medicines, etc.

[0096] Biological Deposit Certificate

[0097] Neurospora intermedia MN134912 is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code 510070, deposit date: November 8, 2024, and the deposit number is GDMCC No.65441. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0099] Figure 1 This is a growth observation diagram of strain MN134912 on a MEA plate in Example 1;

[0100] Figure 2 is a microscopic observation of the mycelium of the strain MN134912 in Example 1;

[0101] Figure 3 is the phylogenetic tree of strain MN134912 in Example 1;

[0102] Figure 4 The fermentation broth obtained after the fermentation culture of strain MN134912 in Example 4 is completed;

[0103] Figure 5 It is the wet base obtained after the fermentation culture of strain MN134912 in Example 4;

[0104] Figure 6 This is a microscopic observation of the mycelium of the strain MN134912 after the fermentation culture in Example 4; the scale bar is 200 μm;

[0105] Figure 7 is a chromatogram of L-ergothioneine standard working solution;

[0106] Figure 8 is a standard curve graph of L-ergothioneine;

[0107] Fig. 9 It is the L-ergothioneine detection result corresponding to the fermentation medium optimized in Example 5. DETAILED DESCRIPTION

[0108] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0109] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0110] Example 1 Isolation and identification of Neurospora intermedius strain MN134912

[0111] 1. Strain source

[0112] 1.1 Sample collection: Traditional Chinese medicinal materials were collected from Hainan Province in April 2020.

[0113] 1.2 Separation process:

[0114] Weigh 2 g of sample, add 20 mL of 0.7% sodium carboxymethyl cellulose suspension (CMC) or other buffer, and shake on a shaker for 30 min (150 rpm) to obtain 10 -1 g / mL sample diluent, pipette 1mL sample diluent into 9mL normal saline, mix well to get 10 -2 g / mL sample diluent, and then serially diluted to 10 -3 ~10 -4 g / mL, take 10 -2 g / mL, 10 -3 The sample dilutions of two gradients of 1:1 and 2:1 were spread on potato dextrose agar plates (PDA plates, formula: 200 g potato extract, 20 g glucose, 15 g agar, 1 L water), and inverted in a constant temperature incubator at 25°C for 3-7 days.

[0115] 1.3 Purification process:

[0116] The mycelium end step-by-step transplantation method was used for purification. After the colonies were formed on the plate, the mycelium at the edge of the single colony was picked up and placed on the PDA plate, and continued to be cultured at 25°C until pure colonies were obtained, and the obtained colonies were stored at 4°C.

[0117] 2. Strain Identification

[0118] 2.1 Morphological characteristics of strain MN134912

[0119] The strain MN134912 was inoculated into MEA medium (Huankai Biotechnology) and cultured at 28°C for 4 days. The strain was identified based on growth rate, hyphae shape, colony color, number and shape of small conidia and large conidia, spore-forming cells, shape and presence of thick-walled spores, fruiting body type, etc.

[0120] Colony culture characteristics: After 4 days of culture, the colony diameter is 4-6cm. The colony grows on the MEA medium in a spreading manner, with luxuriant hyphae and irregular colonies. The conidia are light yellow, the surface of the substrate is white, and the substrate turns orange after being left for a long time. The culture plate is as follows: Figure 1 As shown;

[0121] Microscopic examination of hyphae morphology: Asci are clustered or scattered, asci are cylindrical, with short stalks. Hyphae length: 500-3000μm, hyphae diameter 1-10μm. Conidia are orange-yellow, in clusters, spherical to ovoid. Figure 2 shown.

[0122] 2.2 Molecular biological identification of strain MN134912

[0123] 2.2.1 ITS sequence analysis

[0124] 2.2.1.1. DNA template preparation

[0125] Pick an appropriate amount of bacteria from the surface of the colony cultured on the PDA plate into a PCR tube containing 50 μL of lysis solution, put it into a PCR instrument (Dongshenglong ETC811), heat it at 95℃ for 30 minutes, and transfer it to -20℃ for freezing for 15 minutes. After taking it out, put it into a centrifuge (SIGMA3K15) at 3000rpm for 2 minutes to allow the cell fragments and lysis solution to settle to the bottom of the PCR tube, and the supernatant is basically clear. At this time, the fungal genetic material is dissolved in the supernatant.

[0126] 2.2.1.2. PCR system preparation

[0127] Prepare 25 μL of PCR system in a PCR tube according to the following requirements: ultrapure sterile water, 10.8 μL; 2×PCR Mix (Beijing Qingke Biotechnology), 12 μL; dimethyl sulfoxide (DMSO), 1 μL; 10 μmol / L forward primer solution, 0.6 μL; 10 μmol / L reverse primer solution, 0.6 μL. The primer sequences are as follows:

[0128] Table 1. Primer sequences

[0129]

[0130] 2.2.1.3. PCR amplification reaction

[0131] In the PCR tube, add 2 μL of supernatant and the prepared PCR system, and place it in a PCR instrument (Dongshenglong ETC811) for PCR amplification. The PCR reaction conditions are: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 40 s, 54℃ annealing for 30 s, 72℃ amplification for 1 min, 37 cycles; 72℃ for another 10 min. After amplification, the PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing using a sequencer (abi3730XL).

[0132] 2.2.1.4. Sequences obtained by PCR

[0133] ITS sequence:

[0134]

[0135] (See SEQ ID NO.3). The above sequences were compared using the NCBI online comparison system (blast) to compare the gene sequences. The comparison database was Nucleotide collection (nr / nt). The most similar comparison results are shown in the following table:

[0136] Table 2. ITS sequence alignment results

[0137] Gene Accession Species name Identity / % Coverage / % E-value ITS OQ745719.1 Neurospora intermedia 100 100 0.0

[0138] The steps of whole genome evolution tree analysis are as follows: collect the whole genome sequence of Neurospora flora from NCBI, use Mashtree based on the mash algorithm to construct the whole genome evolution tree of MN134912, the results are as follows Figure 3 Shown

[0139] Based on morphological characteristics and ITS sequence analysis, strain MN134912 is Neurospora intermedia. The strain was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on November 8, 2024, with the deposit address on the fifth floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, zip code 510070, and the deposit number is GDMCC No. 65441.

[0140] Example 2 Determination of mycelial protein

[0141] Activate strain MN134912: inoculate strain MN134912 onto MEA plate culture medium (Huankai Biotechnology) and culture at a constant temperature of 28°C for 48-96h.

[0142] The activated strain was inoculated into a 250 mL tri-concave conical flask containing 50 mL of the primary screening medium and cultured at 28°C and 200 rpm for 48 h. The primary screening medium formula is: glucose 30 g / L, yeast extract 30 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.2 g / L, and the balance is water. It was sterilized by high pressure steam at 115°C for 30 min before use.

[0143] After 48 hours of cultivation, the filter cake was filtered with a vacuum filtration device, and the filter cake was fully washed and dried in an electric blast drying oven at 60°C. The mass of the dried filter cake was accurately weighed and the biomass concentration was calculated. Then, the crude protein of mycelium was determined by the Kjeldahl method in accordance with GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food. The results are as follows:

[0144] Table 3. Mycelium crude protein determination results

[0145] Mycelium morphology Filter cake color Filter cake texture Dry weight g / 50mL Biomass% Mycelial protein content% Filamentous Off-white Flexible 0.62 1.24 56.45

[0146] Biomass = mycelium dry weight / medium volume × 100%;

[0147] Mycelial protein content = crude protein mass / total mass of mycelium × 100%.

[0148] Therefore, the use of strain MN134912 to ferment and prepare mycelial protein can obtain filamentous mycelial morphology, off-white filter cake, elastic texture of the filter cake, dry weight of 0.62 g / 50 mL, biomass of 1.24%, and mycelial protein content of 56.45%.

[0149] Example 3 Study on the Optimal Culture Conditions

[0150] 1. Strain activation

[0151] The strain MN134912 was inoculated onto MEA plate culture medium (Huankai Biotechnology) and cultured at 28°C for 72 h.

[0152] 2. Shake flask culture

[0153] The activated strain MN134912 was inoculated into a 250 mL three-concave conical flask containing 50 mL of seed culture medium by punching, and cultured at 28°C and 200 rpm for 36 to 48 hours. The seed culture medium formula is shown in the following table, and it was sterilized by high pressure steam at 115°C for 30 minutes before use.

[0154] Table 4. Seed culture medium formula

[0155]

[0156]

[0157] 3. Research on the optimal culture temperature

[0158] 2. Inoculate 50 mL of seed culture solution obtained from the shake flask culture into 4 2 L three-concave conical flasks containing 1 L of seed culture medium (see Table 4), and culture them in incubators at 28°C, 32°C, 37°C, and 40°C for 24 h.

[0159] After the cultivation is completed, the filter cake is obtained by filtration with a vacuum filtration device, and the obtained filter cake is fully washed, and the mass of the filter cake is accurately weighed to obtain the wet weight, and then placed in an electric heating blast drying oven at 60°C for drying, and the mass of the dried filter cake is accurately weighed, and then the mycelial protein content (crude protein mass / total mass of bacteria × 100%) is determined by the Kjeldahl nitrogen determination method in accordance with GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food. The results of the determination are as follows:

[0160] Table 5. Results of determination of optimum culture temperature

[0161] Measurement index / temperature 28℃ 32℃ 37℃ 40℃ Wet weight / g 91.87 59.87 53.38 38.51 Dry weight / g / L 10.96 8.82 8.5 6.01 Protein content / % 58.44 61.93 58.08 57.67

[0162] It can be concluded from the above table that strain MN134912 can grow at a temperature of 28°C-40°C, indicating that the strain MN134912 has a certain temperature tolerance and can be cultured at a high temperature of 40°C, so that the strain MN134912 can adapt well in a wider temperature range, thereby ensuring that it can be scaled up for fermentation production process.

[0163] Furthermore, in the above table, the highest protein content of 61.93% can be obtained when cultured at 32°C, while the highest dry weight of 10.96 g / L can be obtained when cultured at 28°C, so the most suitable culture temperature for MN134912 is 28-32°C.

[0164] 4. Study on the Optimal Culture pH

[0165] 2. Inoculate 50 mL of seed culture solution obtained from shake flask culture into 4 2 L tri-conical flasks containing 1 L of seed culture medium (see Table 4), then adjust the pH of the culture medium to five gradients of 4, 5, 6, 7, and 8 with phosphoric acid and NaOH, respectively, and culture in a 28 °C incubator for 24 h.

[0166] After the culture was completed, the hyphae length, number of branches, and number of spores were observed under a microscope, and then the wet weight / g, dry weight / g / L, and protein content / % were determined according to the method in "3. Study on the Optimal Culture Temperature". The results are as follows:

[0167] Table 6. Results of determination of optimum culture pH

[0168] Determination index / pH 4 5 6 7 8 Wet weight / g 107.49 98.81 81.85 89.98 73.23 Dry weight / g / L 9.54 9.8 9.4 10.05 10 Protein content / % 52.23 53.57 54.69 54.18 52.15 Mycelium length short middle long long long Fork many many few few few Spore count many many none none none

[0169] It can be concluded from the above table that strain MN134912 can grow at pH 4-8, indicating that the strain MN134912 has a certain pH tolerance and good adaptability in a wider pH range, thereby ensuring that it can be scaled up for fermentation production process.

[0170] Furthermore, it can be seen from the above table that strain MN134912 has longer hyphae, less bifurcation and less spore production under the culture condition of increased pH. And its dry weight and protein are up to 10.05 g / L and 54.69% respectively in the range of pH 6-7, so the most suitable pH for MN134912 is 6-7.

[0171] 5. Study on the optimal concentration of metal ions for culture

[0172] 50mL of seed culture solution obtained in 2. Shake flask culture was inoculated into 2L three-concave conical flasks containing 1L seed culture medium (see Table 4), and an orthogonal experiment was designed according to three levels and three factors. The potassium dihydrogen phosphate, magnesium sulfate and calcium chloride in the seed culture medium were set at three different concentration levels, namely high concentration, medium concentration and low concentration. The specific formula design and results are shown in the following table. Place in a 28℃ incubator and culture for 24h. After the culture, the protein content was determined according to the method in "3. Study on the optimal culture temperature".

[0173] Table 7. Results of determination of optimal culture metal ion concentrations

[0174]

[0175]

[0176] Among them, K1, K2, K3 represent the sum of indicators at each level of each factor, and R represents the range, which is equal to the maximum average yield - the minimum average yield. For example, K1(KH2PO4) = 57.53 + 61.25 + 60.29; R(KH2PO4) = 59.69-57.88.

[0177] It can be seen from the above table that according to the results of orthogonal test range analysis, the optimal ion formula of high protein MN134912 is low concentration KH2PO4, medium concentration MgSO4, and low concentration CaCl2.

[0178] Example 4 50 L scale-up production process of strain MN134912

[0179] 1. Bacteria activation

[0180] The strain MN134912 was inoculated onto MEA plate culture medium (Huankai Biotechnology) and cultured at 28°C for 72 h.

[0181] 2. Seed cultivation

[0182] 2.1 Primary seed culture

[0183] The activated MN134912 strain was selected and inoculated into a 250 mL three-concave conical flask containing 50 mL of the primary seed culture medium, and cultured at 28°C and 200 rpm for 24 hours. The formula of the primary seed culture medium is shown in the following table, and it was sterilized by high-pressure steam at 115°C for 30 minutes before use.

[0184] Table 8, Primary Seed Culture Medium Formula

[0185]

[0186]

[0187] 2.2 Secondary seed culture

[0188] The obtained primary seed liquid was transferred to a 3L tri-conical flask containing 1L secondary seed culture medium at an inoculation rate of 5v / v%, and cultured at 28°C for 24h at a shaking speed of 200rpm. The difference between the secondary seed culture medium and the primary seed culture medium is that the yeast extract concentration is reduced to 10g / L.

[0189] 3. 50L fermentation culture to produce mycelial protein

[0190] The obtained secondary seed liquid was transferred to a production fermenter filled with 50L fermentation medium at a 5% inoculation rate, stirred and cultured at 50-200rpm, the pH of the fermentation process was controlled to 6-7, fermented at 28-32°C, dissolved oxygen linkage controlled to 20-50%, ventilation 15-300L / min, and fermented for 24h. The fermentation medium formula (solvent is deionized water) is as shown in the following table, and it was sterilized by high-pressure steam at 115°C for 30min before use.

[0191] Table 9, 50L fermentation medium

[0192] Element Fermentation medium Corn starch saccharification liquid 30g / L Citric acid 1g / L Potassium dihydrogen phosphate 3g / L Ammonium sulfate 5g / L Magnesium sulfate heptahydrate 0.3g / L Calcium chloride 0.01g / L Manganese sulfate monohydrate 1.7mg / L Ferrous Sulfate Heptahydrate 5mg / L Zinc chloride 1.7mg / L Vitamin B solution (commercially available) 1mL / L

[0193] After fermentation, microscopic examination and protein detection were performed. The results were as follows Figure 4-6 The results of microscopic examination were as follows: the filaments were excellent, the filter cake was light flesh color, tough, and had no obvious odor; therefore, the tasting result was good. The protein test result was 55.4%, the yield was 52g / L in wet weight, and 9.35g / L in dry weight (wet weight / g, dry weight / g / L, and protein content / %) were determined according to the method in "3. Study on the Optimal Culture Temperature" in Example 3.)

[0194] Example 5 Detection of ergothioneine in hyphae protein of strain MN134912

[0195] 1. Detection of key synthetic genes of ergothioneine

[0196] Ergothioneine key synthetic gene is EGT-1 and EGT-2 (NCU11365, NCU04343), the protein sequence of the above two ergothioneine synthetic genes is collected to build ergothioneine synthetic gene database, by blastp software, the protein sequence of bacterial strain MN134912 is compared with ergothioneine synthetic gene database, with sequence similarity ≥ 60%, sequence coverage ≥ 60%, as screening criteria, the ergothioneine synthetic gene in target strain is obtained.Comparison result is as follows, and it is illustrated that bacterial strain MN134912 has ergothioneine key synthetic gene EGT-1 and EGT-2.

[0197] Table 10 Comparison results

[0198] Sequence number protein Reference sequence % identity Coverage % Length (amino acids) MN134912_002091-T1 EGT-1 NCU04343 98.29 100 877 MN134912_003116-T1 EGT-2 NCU11365 97.04 100 473

[0199] 2. Detection of ergothioneine in mycelial protein

[0200] The method for detecting ergothioneine content is in accordance with the agricultural industry standard NY / T 3872-2021, as follows:

[0201] 1) Sample preparation and extraction

[0202] Weigh 1g of sample (accurate to 0.01g) and place it in a 50mL centrifuge tube. Add 40.00mL of 70v / v% methanol aqueous solution accurately. Ultrasonic extraction was performed in an ultrasonic cleaner for 30min. Centrifuge at 4000r / min for 5min. Take 2mL of the extract and blow it to near dryness. Redissolve it with 2mL of methanol solution containing 0.1v / v% formic acid. After filtering through a 0.22μm filter membrane, take 1mL of the filtrate for determination by ultra-performance liquid chromatography.

[0203] 2) Chromatographic reference conditions

[0204] Chromatographic column: HILIC column (2.1mm×100mm, 1.7μm), or chromatographic column of equivalent specifications;

[0205] Mobile phase: acetonitrile solution containing 0.1 v / v% formic acid-water solution containing 0.1 v / v% formic acid;

[0206] Elution method: the mobile phase contains 0.1v / v% formic acid in acetonitrile solution: 0.1v / v% formic acid in water solution at a volume ratio of 50%:50% for isocratic elution;

[0207] Flow rate: 0.4 mL / min;

[0208] Column temperature: 40°C;

[0209] Wavelength: 262nm;

[0210] Injection volume: 3 μL.

[0211] 3) Drawing of standard curve

[0212] Use L-ergothioneine series standard working solution, measure with reference to the above-mentioned chromatographic conditions, take the mass concentration of standard working solution as abscissa, peak area as ordinate to draw the standard curve of L-ergothioneine. The chromatogram of L-ergothioneine standard working solution is shown in Figure 7 The standard curve of L-ergothioneine is as follows: Figure 8 .

[0213] 2.1. Culture of strain MN134912

[0214] 2.1.1 Bacterial Activation

[0215] The strain MN134912 was inoculated into MEA plate culture medium (Huankai Biotechnology) and cultured at 28°C for 72 hours.

[0216] 2.1.2. Detection of ergothioneine in fermented mycelium of MN134912

[0217] The activated strain MN134912 was inoculated into a fermenter containing 50 L of culture medium, and the fermentation method was the same as in Example 4, except that the fermentation medium used was the medium listed in Table 11. The culture medium was sterilized by high pressure steam at 115° C. for 30 min before use.

[0218] Table 11. Optimized culture medium

[0219] Formula Materials Culture medium (g / L) glucose 30 Yeast extract powder 10 Potassium dihydrogen phosphate 1 Magnesium sulfate heptahydrate 0.5 Calcium chloride 0.2 Ferrous Sulfate 1 Histidine 1

[0220] After fermentation culture terminates, the mycelium obtained is used as sample, 6g samples (accurate to 0.01g) are weighed, it is placed in 50mL centrifuge tube, 100ml pure water is added to mix, homogenizer 8000-10000rpm is used to homogenize 15min, mycelia homogenate liquid is obtained.Evenly take 10mL mycelia homogenate liquid and add 70v / v% methanol aqueous solution, after ultrasonic cleaner ultrasonic extraction 30min, 4000r / min, centrifugal 5min, take 2mL extracting solution and be blown to nearly dry, with 2mL containing 0.1v / v% formic acid methanol solution redissolve, after crossing 0.22 μm filter membrane, take 1mL filtrate and detect according to aforementioned thioneine content detection method.

[0221] The results of the optimized fermentation medium are as follows Fig. 9 As shown, in the mycelium sample group of strain MN134912, its peak time is consistent with that of the standard, peaking at 9 minutes, and the peak area of ​​ergothioneine is 796666. Figure 8 In the standard curve, 62.11mg / L of ergothioneine concentration in sample is calculated, and 1630.3μg / g dry weight (calculating formula is ergothioneine quality / mycelium dry weight × 100%) is calculated in mycelium through conversion. It can be drawn from the above that the mycelium of MN134912 of the present invention is rich in ergothioneine, significantly higher than the ergothioneine content in other natural fungi in the Neurospora genus of existing report, such as ergothioneine content 466~850μg / g dry weight (Genghof, 1970, Biosynthesis ofErgothioneine and Hercynine by Fungi and Actinomycetales DOROTHY S.GENGHOF) reported, higher than the dry weight (Dubost et al. (2007)) of ergothioneine content 210ng / mg~2600ng / mg (oyster mushroom) in other fungi.

[0222] Example 6 Detection of nutritional components in mycelial protein of strain MN134912

[0223] The 50L fermentation culture produced mycelial protein in Example 4 was used as a sample for nutrient component detection. The nutrient component detection method was: cholesterol GB5009.128-2016; unsaturated fatty acids GB5009.168-2016; dietary fiber GB5009.88-2023. The test results are as follows:

[0224] Table 12. Nutritional content of mycelial protein

[0225]

[0226] Table 14 continued, nutritional content

[0227]

[0228] It can be seen from the above table that, in terms of cholesterol content, the cholesterol content in the dry and wet basis of mycelial protein or mycelium of the strain MN134912 of the present invention is very low, much lower than that of beef and chicken.

[0229] In terms of total monounsaturated fat content, the dry basis and wet basis total monounsaturated fat content of the mycelial protein or mycelium of the strain MN134912 of the present invention is lower than that of beef and chicken.

[0230] In terms of total polyunsaturated fat content, the mycelial protein or mycelium of the strain MN134912 of the present invention has a higher total polyunsaturated fat content on a dry basis and a wet basis than beef and chicken.

[0231] In the total dietary fiber content, the dry basis total dietary fiber content of mycelial protein or mycelium of the strain MN134912 of the present invention is 35.68%, while beef and chicken do not contain total dietary fiber.

[0232] In terms of protein content, the protein content in the dry basis of mycelial protein or mycelium of the strain MN134912 of the present invention is better than that of chicken and beef.

[0233] The above shows that the mycelial protein or mycelium of the strain MN134912 of the present invention is healthier when applied in the food field. Compared with chicken and beef, it has low cholesterol, appropriate unsaturated fatty acid content, high total dietary fiber and protein content, and its nutritional components are equivalent to high-quality meat.

[0234] In addition, the mycelium of the strain MN134912 of the present invention is rich in amino acids and trace elements required by the human body, and has the advantages of high energy and low carbohydrates, in addition to the nutrients listed in Table 14.

[0235] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Neurospora intermedia, characterized in that Any one of the following A1) to A4): A1), a strain whose ITS sequence is at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the sequence shown in SEQ ID NO: 3; A2), Neurospora intermedia MN134912, deposited in Guangdong Microbiological Culture Collection Center, with the deposit number of GDMCC No.65441; A3) a strain having at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identity with the ITS sequence of the strain described in A2); A4), a strain whose genome has an average nucleotide identity of at least 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% with the genome of any of the strains described in A1) to A3).

2. The Neurospora intermedia according to claim 1, characterized in that The Neurospora intermedia contains an ergothioneine synthesis gene; Optionally, the Neurospora intermedia comprises a sequence that is at least 95%, 95.5%, 96.5%, 97%, 97.5%, 98%, 98.29%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or 100% identical to the sequence of at least one of the ergothioneine synthesis genes EGT-1 and EGT-2.

3. A microbial agent, characterized in that: It comprises the Neurospora intermedia according to claim 1 or 2, its inactivated product, its culture, its fermentation product, and / or its extract.

4. A mycelial protein or mycelium, characterized in that: It is prepared by the Neurospora intermedia according to claim 1 or 2 or the microbial agent according to claim 3; Preferably, the hyphae length of the hyphae protein or mycelium is 500-3000 μm, and / or the hyphae diameter is 1-10 μm; Preferably, the hyphae protein or mycelium contains at least one of cholesterol, unsaturated fatty acids, dietary fiber, protein, ergothioneine, amino acids and trace elements; Preferably, the protein content is higher than 45% based on the dry weight of mycelial protein or mycelium; Preferably, the protein content is 50%-65% based on the dry weight of mycelial protein or mycelium; Preferably, the dietary fiber content is higher than 25% based on the dry weight of mycelial protein or mycelium; Preferably, the dietary fiber content is 30%-40% based on the dry weight of mycelial protein or mycelium; Preferably, based on the dry weight of the mycelial protein or mycelium, the ergothioneine content of the mycelial protein or mycelium is 500-2000 μg / g dry weight; Preferably, the unsaturated fatty acid comprises at least one of total monounsaturated fatty acids and total polyunsaturated fatty acids; And / or, the dietary fiber includes at least one of soluble dietary fiber and insoluble dietary fiber; And / or, the trace elements include at least one of calcium, iron, sodium and potassium; And / or, the amino acids include but are not limited to tryptophan.

5. Use of Neurospora intermedia in producing mycelial protein or mycelium, characterized in that: Fermentation culture is carried out using the Neurospora intermedia described in claim 1 or 2 or the microbial agent described in claim 3.

6. A method for producing mycelial protein or mycelium, characterized in that: The method comprises the following steps: utilizing Neurospora intermedia to carry out fermentation culture, separating mycelium from the fermentation product, and obtaining mycelium protein; Preferably, the Neurospora intermedia is the Neurospora intermedia described in claim 1 or 2 or the microbial agent described in claim 3; Preferably, before fermentation culture, the method further comprises the steps of activating the Neurospora intermedia, seed culture and / or amplifying culture; Preferably, the fermentation culture includes at least one of liquid fermentation and solid fermentation; preferably, it is liquid fermentation; Preferably, the pH of the medium used in the fermentation culture is 4-8, preferably, the pH is 6-7; And / or, the fermentation culture temperature is 28-40°C, preferably 28-32°C; And / or, the fermentation medium used in the fermentation culture has a K + The concentration is 1-10g / L, Mg 2+ The concentration is 0.3-5g / L, Ca 2+ The concentration is 0.01-1g / L, preferably, K + The concentration is 1-5g / L, Mg 2+ The concentration is 0.3-2g / L, Ca 2+ Concentration is 0.01-0.5g / L; Preferably, the culture medium also includes adding a nutrient component that promotes the synthesis of ergothioneine; Optionally, the nutritional components include but are not limited to at least one of histidine and ferrous sulfate; Optionally, the concentration of the histidine and ferrous sulfate is 0.1-10 g / L, preferably, the concentration of the histidine and ferrous sulfate is 1 g / L.

7. A mycelial protein or mycelium obtained by the method according to claim 6.

8. Use of Neurospora intermedia according to claim 1 or 2, the microbial agent according to claim 3, or the mycelial protein or mycelium according to any one of claims 4 to 6 in the production of alternative proteins, alternative protein products, ergothioneine, or products containing ergothioneine; Preferably, the alternative protein product includes food, condiment or feed; Preferably, the product containing ergothioneine includes food, medicine or cosmetics; optionally, the food includes health products or functional foods; Preferably, the food includes but is not limited to at least one of meat analogs, plant-based protein meat products, dairy products, beverages, dietary fiber foods, baked products, spreadable products, health products or functional foods; Optionally, the meat analogs include, but are not limited to, meat analogs of livestock, seafood, and poultry; Optionally, the plant-based protein meat products include, but are not limited to, plant-based protein meat products from livestock, seafood, and poultry; Optionally, the livestock includes but is not limited to at least one of pigs, cattle, and sheep; Optionally, the seafood includes but is not limited to at least one of fish, shrimps, crabs, and shellfish; Optionally, the plant-based protein meat products include, but are not limited to, plant-based protein meat products derived from soy protein, pea protein, peanut protein, and wheat protein; Optionally, the meat analogs and plant-based protein meat products include but are not limited to meat sausages, minced meat, meat balls, meat patties, meat-like cuts, and meat-like sliced ​​products such as ham and bacon; Optionally, the dietary fiber food includes cereal product substitutes and fruit and vegetable product substitutes; Optionally, the health product or functional food includes a protein supplement, an amino acid supplement or a dietary fiber supplement; Preferably, the feed includes poultry feed, livestock feed, aquaculture feed or pet feed.

9. A protein food ingredient comprising a. cultured Neurospora intermedia biomass in an amount of at least 3.8% w / w; and b. water in an amount of up to 96.2% w / w; Preferably, the Neurospora intermedia is the Neurospora intermedia according to claim 1 or 2; Preferably, the Neurospora intermedia biomass is the Neurospora intermedia hyphal protein or mycelium according to any one of claims 4 to 6; Preferably, the protein food ingredient comprises 5% w / w-55% w / w of the cultured Neurospora intermedia biomass and 95% w / w-45% w / w of water; Preferably, the protein food ingredient comprises 5% w / w-15% w / w of the cultured Neurospora intermedia biomass and 95% w / w-85% w / w of water; Preferably, the protein food ingredient comprises 90% w / w-99% w / w of the cultured Neurospora intermedia biomass and 1% w / w-10% w / w of water; Preferably, the protein food ingredient further comprises carbohydrates, fat, protein, fiber or any combination thereof; Preferably, the carbohydrate comprises glucose, fructose, galactose, maltose, sucrose, lactose, starch or any combination thereof; Preferably, the carbohydrate comprises white sugar, brown sugar, black sugar, agar, pectin, gum arabic, molasses, honey, cane sugar, wheat starch, corn starch, potato starch, food starch, dextrose or any combination thereof; Preferably, the fat comprises vegetable oil, animal oil or any combination thereof; Preferably, the fat comprises safflower oil, sunflower oil, olive oil, coconut oil, sunflower oil, safflower oil, rapeseed oil, canola oil, vegetable glycerin, soy lecithin or any combination thereof; Preferably, the protein comprises plant protein, animal protein, microbial protein or any combination thereof; Preferably, the protein comprises texturized pea protein, texturized wheat protein, texturized soy protein, soy protein, wheat protein, pea protein, rice protein, potato protein, albumin or any combination thereof; Preferably, the fiber comprises pea fiber, oat bran, other plant fiber, bamboo fiber, cellulose, citrus fiber, soy fiber or any combination thereof; Preferably, the protein food ingredient further comprises soybeans, lentils, lentil flour, soybean flour, pea flour, yeast extract, nutritional yeast (fixed dry yeast), mushroom powder, white Agaricus bisporus powder, shiitake mushroom powder, chickpeas, isolated oat products, isolated pea products, fermented rice extract, kelp extract, algae, silicon dioxide, vitamins, dry yeast, citrus extract, beet pulp, beet juice, beet juice extract, turmeric, mushroom extract, shiitake mushroom stems, shiitake mushrooms, white Agaricus bisporus, tofu, soybean hydrolyzate, maltodextrin, sodium ascorbate, calcium carbonate, yeast extract, seaweed, spices, sweeteners, natural flavoring agents or any combination thereof.

10. An edible composition, characterized in that The method comprises the mycelial protein or mycelium according to any one of claims 4 to 6, or the protein food ingredient according to claim 9, and a food additive; the amount of the mycelial protein or mycelium or the protein food ingredient added is ≥0.01wt% of the total amount.

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