A strain of Neurospora intermedia and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a strain of Neurospora intermedia and its application. Neurospora intermedia , whose strain number is OK, has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 10, 2024, with a deposit number of CGMCC No.41150. The mycelium of Neurospora OK described in the present invention contains high protein, can produce high cellulase, and does not produce mycotoxins. The fungus can be cultivated using by-products of soy product processing such as yellow pulp water and bean dregs, and the obtained fungal protein can be used as an alternative protein raw material for the production of chicken nuggets and vegetarian patties, saving costs and increasing environmental benefits, and therefore has broad application prospects in food processing.

Description

Technical Field

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

[0002] Neurospora intermedia can grow rapidly on cheap carbon sources such as molasses and straw, and can effectively decompose complex carbohydrates and proteins and produce a large amount of protein and cellulase during the cultivation process.

[0003] A large number of studies have found that the protein content of fungal protein is as high as 40%~60%, which is 10%~20% higher than soybeans and more than 20% higher than meat, fish and cheese. In addition, as a substitute for plant and animal protein, fungal protein has the advantages of high production efficiency, a wide range of raw material sources, and is not restricted by regional, seasonal, and climatic conditions. Neurospora intermedia fungal protein has a meaty fiber structure and can form the desired meat texture without complex processing. It has the potential for application as a substitute for plant and animal protein. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a strain of Neurospora intermedia in view of the deficiencies of the prior art.

[0005] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned Neurospora intermedia.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A strain of Neurospora intermedia, named Neurospora intermedia , the strain number is OK, and it was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (abbreviation: CGMCC) on April 10, 2024. The deposit number is CGMCC No. 41150, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Among them, the nucleotide sequence of ITS of the Neurospora intermedia is shown as SEQ ID No.1.

[0009] Among them, the Neurospora intermedia has a strong carbohydrate metabolism ability and can utilize a variety of carbon sources to meet growth metabolism; it has good metabolic growth ability and cell regeneration ability, and can withstand certain harsh environments; and it does not have the ability to synthesize fungal toxins, and the mycelium protein content is higher than 40%.

[0010] The use of the above-mentioned Neurospora intermedia in the production of cellulase is also within the scope of protection of the present invention.

[0011] A method for producing cellulase using the Neurospora intermedia, specifically, inoculating a Neurospora intermedia OK spore suspension into a fermentation medium for fermentation culture, and centrifuging to obtain a supernatant containing cellulase.

[0012] Wherein, the concentration of the spore suspension is 10 4 ~10 7 spores / mL, the inoculum amount in the fermentation medium is 0.5~3% v / v; the preferred spore suspension concentration is 10 6 spores / mL, and the inoculum size was 1% v / v.

[0013] The fermentation culture is carried out under the conditions of 24-37° C. and 120-180 rpm for 120-168 h; the fermentation medium is yellow pulp water containing 5-10% dregs of soybean dregs, and the preferred amount of dregs added is 8 wt% or 10 wt%.

[0014] Specifically, the yellow pulp water containing 5-10% bean dregs needs to be sterilized before use, and the sterilization temperature is 100-115° C. and the sterilization time is 15-30 min.

[0015] Preferably, the culture conditions are 35° C. and 150 rpm for 120 h.

[0016] The use of the above-mentioned Neurospora intermedia in the production of fungal protein is also within the scope of protection of the present invention.

[0017] A method for producing fungal protein using the Neurospora intermedia, specifically, inoculating a suspension of Neurospora intermedia spores into a fermentation medium for fermentation culture, and then washing and drying to obtain the fungal protein.

[0018] Wherein, the concentration of the spore suspension is 10 4 ~10 7 spores / mL, the inoculum amount in the fermentation medium is 0.5-3% v / v; preferably, the concentration of the spore suspension is 10 6 spores / mL, and the inoculum size was 1% v / v.

[0019] Wherein, the fermentation medium is yellow pulp water containing 5-10% bean dregs; preferably, it is yellow pulp water containing 10% bean dregs.

[0020] Specifically, the yellow pulp water containing 5-10% bean dregs needs to be sterilized before use, and the sterilization temperature is 100-115° C. and the sterilization time is 15-30 min.

[0021] Wherein, the drying is carried out by any one of hot air drying, freeze drying and microwave drying.

[0022] The fermentation culture strategy adopts low-frequency ultrasound combined with single batch fermentation culture or low-frequency ultrasound combined with repeated batch culture; preferably, low-frequency ultrasound combined with repeated batch culture.

[0023] The fermentation culture strategy adopts low-frequency ultrasound combined with repeated batch culture, and the specific steps are as follows:

[0024] (1) Inoculate a spore suspension of Neurospora intermedia into a fermentation medium for fermentation. When the reducing sugar content is lower than 0.5 g / L, discharge 10-70% v / v of the fermentation liquid and add an equal amount of fresh fermentation medium.

[0025] (2) Continue culturing. When the reducing sugar content is lower than 0.5 g / L, continue to discharge 10-70% v / v of the fermentation liquid and add an equal amount of fresh fermentation medium.

[0026] (3) Repeat step (2) and terminate the fermentation reaction when the yield of the fermented fungal protein is less than 50% of the yield of the previous batch of fermentation.

[0027] Specifically, in steps (1) and (2), preferably, 50% of the fermentation liquid is discharged.

[0028] Specifically, in the step (3), the repetition is repeated 1 to 4 times, preferably 3 times.

[0029] Specifically, the fermentation conditions of the fermentation culture are: 24~37°C, 120~180 rpm; ultrasonic treatment is performed during the fermentation, and the ultrasonic conditions are: fixed frequency of 20~40 kHz, pulse mode of 3~6 s on and 4~10 s off, ultrasonic density of 0~165.53 W / L, ultrasonic time of 4~16 min, and ultrasonic interval of 6~24 h.

[0030] Preferably, the fermentation conditions of the fermentation culture are: 35°C, 150 rpm; the ultrasonic conditions are: fixed frequency of 23kHz, pulse mode of 5 s on and 5 s off, ultrasonic density of 116.20 W / L, ultrasonic time of 8 min, and ultrasonic interval of 12 h.

[0031] Among them, the above-mentioned method for producing fungal protein can significantly reduce the chemical oxygen demand (COD) and biological oxygen demand (BOD) in yellow pulp water containing 5-10% bean dregs, reducing the burden on the environment.

[0032] The fungal protein prepared by the method for producing fungal protein using Neurospora intermedia is also within the scope of protection of the present invention.

[0033] The use of the above-mentioned Neurospora intermedia or the above-mentioned fungal protein in the preparation of fungal protein food is also within the scope of protection of the present invention.

[0034] In some embodiments of the present invention, the fungal protein food is fried chicken or vegetarian meat patties.

[0035] Beneficial effects:

[0036] 1. The present invention isolates and screens a strain of Neurospora intermedia OK from red fungus tofu. The mycelium of the strain has a high protein content, can produce a high amount of cellulase, and does not produce mycotoxins.

[0037] 2. The present invention adopts low-frequency ultrasound combined with repeated batch culture to prepare fungal protein using Neurospora intermedia OK, and significantly improves the yield and production efficiency of fungal protein.

[0038] 3. The present invention turns waste into treasure by using bean dregs, a waste in the food processing process, as a carbon source and yellow pulp water as a fermentation medium to ferment and produce fungal protein. In the process of producing fungal protein, the chemical oxygen demand (COD) and biological oxygen demand (BOD) in the yellow pulp water containing bean dregs can be significantly reduced, thereby reducing pollution to the environment.

[0039] 4. The Neurospora intermedia OK fungal protein of the present invention can be used as a substitute protein for producing fungal protein foods, and can simulate the texture and taste of meat products to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0041] Figure 1 Neurospora intermedia OK GO functional annotation;

[0042] Figure 2 Neurospora intermedia OK KEGG functional annotation;

[0043] Figure 3 Neurospora intermedia OK CAZy functional annotation;

[0044] Figure 4 Effects of different amounts of okara added on the yield of Neurospora intermedia OK fungal protein in yellow pulp water;

[0045] Figure 5 Effect of ultrasonic density on the yield of Neurospora intermedius OK fungal protein in yellow pulp water containing 10% soybean dregs;

[0046] Figure 6 Effect of ultrasound time on the yield of Neurospora intermedius OK fungal protein in yellow pulp water containing 10% soybean dregs;

[0047] Figure 7 Effect of ultrasound interval on the yield of Neurospora intermedius OK fungal protein in yellow pulp water containing 10% soybean dregs;

[0048] Figure 8 Sensory evaluation radar chart of vegetarian meat patties with added Neurospora intermedius OK mycelium. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0050] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0051] Example 1 Isolation, identification and whole genome sequencing of strains

[0052] 1. Isolation of bacterial strains

[0053] Red fungus tofu was collected from Wuzhou, China on July 31, 2023. The marginal hyphae of single colonies with different morphologies were picked and inoculated on PDA medium plates. They were repeatedly separated and purified for more than 3 generations and numbered. The purified hyphae were inoculated on PDA slants, cultured in a 28°C incubator for 48 h, and then stored in a 4°C refrigerator for further identification.

[0054] 2. Identification of strains using molecular biology techniques

[0055] According to the manufacturer's instructions, the genomic DNA of the strain to be identified was extracted using a genome extraction kit (Tiangen Biochemical Technology Co., Ltd., DP302), and the ITS gene fragment of the strain to be identified was amplified using the universal primer pair ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (the sequence is shown in SEQ ID No. 1). The PCR amplification product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were compared with the Genbank database of NCBI, and it was found that the strain with the highest homology to the strain was Neurospora intermedia Neurospora intermedia Therefore, the strain was identified as Neurospora intermedia Neurospora intermedia .

[0056] The identified strains were preserved and classified as: Neurospora intermedia Neurospora intermedia, strain number: OK, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 10, 2024, with the deposit number CGMCC No. 41150, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0057] 3. Whole genome sequencing and analysis of Neurospora intermedius OK

[0058] (1) Whole genome sequencing:

[0059] The purified cells of Neurospora intermedia OK were sent to Meiji Biotechnology Co., Ltd. for sequencing, and Illumina library and PacBio full gene library were constructed. denovo The sequencing sequences were assembled and the genome of the strain was analyzed using bioinformatics analysis. The results showed that the effective chromosome sequence length of Neurospora intermedia OK was 40167057bp, containing 14 Scaffolds, with a GC content of 48.55%, 8544 CDSs in the coding sequence region that can be translated into proteins, 412 rRNAs and 173 tRNAs.

[0060] (2) Annotation results analysis:

[0061] Based on COG database annotation: A total of 2617 genes were annotated in Neurospora intermedia OK, as shown in Table 1. The largest number of genes were carbohydrate transport and metabolism genes, followed by general function prediction genes, translation, ribosomal structure and biogenesis, lipid transport and metabolism, and amino acid transport and metabolism, which indicated that Neurospora intermedia OK had strong carbohydrate metabolism capabilities.

[0062] Table 1 COG functional classification of Neurospora intermedia OK

[0063]

[0064] Based on GO database annotation: It was found that among the 5960 annotated genes, the most genes were related to cellular anatomical entity, cellular process, metabolic process, catalytic activity and binding ( Figure 1 ), indicating that the bacteria has good metabolic growth and cell regeneration capabilities and can withstand certain harsh environments.

[0065] KEGG metabolic pathway annotation: KEGG is a database for genome decoding. Functional analysis of annotated genomes based on KEGG pathways is performed. Figure 2 As shown, a total of 5048 genes were annotated in 48 pathways in 6 branches in Neurospora OK, among which there were 1322, 405, 361 and 357 genes related to global and overview maps, carbohydrate metabolism, signal transduction and transport and catabolism, respectively, which were the 4 most abundant annotations of Neurospora OK.

[0066] Functional annotation of carbohydrate-active enzymes (CAZy): The CAZy database can classify enzymes into five categories according to their functions: glycoside hydrolases (GHs), glycosyltransferases (GTs), oxidoreductases (AAs), carbohydrate esterases (CEs), and polysaccharide lyases (PLs). Figure 3 As shown, the genome of Neurospora intermedia OK has the most genes for glycoside hydrolases (GHs), with 154, followed by oxidoreductases (AAs), with 69. These results indicate that Neurospora intermedia OK is able to utilize a variety of carbon sources to satisfy its growth metabolism.

[0067] The polyketide synthase gene synthetase pathway (PKS), non-ribosomal peptide synthetase pathway (NRPS) and polyketide synthase gene synthetase-non-ribosomal peptide synthetase mixed metabolism (PKS-NRPS) are the key metabolic pathways in the synthesis of most fungal toxins. Anti-SMASH was used to analyze and predict the synthesis of secondary metabolites in the genome, and a total of 19 secondary metabolite gene clusters were predicted in the genome, mainly polyketide synthase (PKS) gene clusters, non-ribosomal peptide synthetase (NRPS) gene clusters and terpenes, as shown in Table 2. BLAST comparison of all gene clusters with known secondary metabolites revealed that although the gene clusters of the PKS pathway and NRPS pathway in the genome of Neurospora intermedia OK were annotated with gene information with relatively high similarity, they were not mycotoxin synthesizing. It can be seen that Neurospora intermedia OK does not have the ability to synthesize mycotoxins. In addition, no mycotoxins were detected from the mycelium of Neurospora intermedia OK.

[0068] Table 2 OK secondary metabolite biosynthesis gene cluster

[0069]

[0070] Example 2 Cellulase production of Neurospora intermedia OK

[0071] 1. Comparison of cellulase production from filamentous fungi from different sources

[0072] Preparation of filamentous fungal spore suspension: The filamentous fungi were inoculated on potato dextrose agar medium and cultured at 28°C for 5 days. The spores were washed off with sterile water to prepare 10 6 The yellow pulp water was used as the fermentation medium and sterilized at 108℃ for 20 min. After cooling, the spore suspension (10 6 The spores / mL were inoculated into yellow slurry water medium at a rate of 1% v / v and cultured at 35°C and 150 rpm for 120 h.

[0073] The fermented yellow pulp water medium was centrifuged at 10000 g for 10 min, and the obtained supernatant was used to determine the cellulase activity. The endoglucanase activity was determined using sodium carboxymethylcellulose as a substrate. Specifically, 1 mL of citric acid buffer (pH 4.8, containing 1% sodium carboxymethylcellulose) was incubated with 0.5 mL of the supernatant and then incubated at 50 °C for 30 min. The release of reducing sugars was determined by the dinitrosalicylic acid method. The unit of exoglucanase activity was defined as the amount of enzyme required to release 1 μmol of glucose equivalent per minute under the conditions. Similarly, the activities of exoglucanase and β-glucosidase were determined using 1 mg / mL of p-nitrophenyl cellobiose glycoside and p-nitrophenyl-β-D-pyranoglucoside as substrates, respectively. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of ρ-nitrophenol per minute under the conditions.

[0074] The filamentous fungi from different sources (Rhizopus oligosporus RT-3, Monascus JM2, Rhizopus oryzae HQ-1, Actinomucor elegans MT-1) were used as control strains to detect the cellulase activity of the corresponding strains. The results of the cellulase activity determination are shown in Table 3. The activities of endoglucanase, exoglucanase and β-glucosidase of Neurospora intermedia OK were higher than those of filamentous fungi from other sources.

[0075] Table 3 Cellulase activities of filamentous fungi from different sources cultured in yellow pulp water

[0076]

[0077] 2. Comparison of endoglucanase activity in yellow pulp water with different amounts of okara added

[0078] The activity of the main cellulase, endoglucanase, in the yellow pulp water inoculated with Neurospora intermedia OK was further analyzed under different amounts of okara addition (6 wt%, 8 wt%, 10 wt% and 12 wt%), and the experimental process was referred to step 1. The results are shown in Table 4. When the amount of okara added was 8 wt%, the activity of endoglucanase in the yellow pulp water after 120 h of culture was the highest, which was 8.23 ​​U / mL.

[0079] Table 4 Endoglucanase activity in yellow pulp water with different addition amounts of okara

[0080]

[0081] Example 3 Optimization of production conditions of Neurospora intermedia OK fungal protein in single batch fermentation and repeated batch fermentation in yellow pulp water

[0082] Saccharification and cultivation is widely considered to be a promising process that can reduce the amount of enzymes used and production costs. This example uses single batch fermentation and repeated batch fermentation to hydrolyze bean dregs using cellulase produced by Neurospora intermedia OK in the culture medium, thereby converting cellulose and hemicellulose into sugars that can be used by Neurospora intermedia OK for growth.

[0083] 1. Effect of different amounts of wet dregs added on fungal protein yield under single batch fermentation

[0084] The yellow pulp water and the yellow pulp water containing 10% bean dregs were used as culture medium, sterilized at 108℃ for 20 min, and after cooling, the spore suspension (10 6 The fungal protein yield in different fermentation broths was determined every 12 h.

[0085] The results are as follows Figure 4 As shown in the figure, the trend of fungal protein production under different wet okara addition amounts (0% and 10%) was compared. When 10 wt% okara was added, the yield of fungal protein increased, and when the culture time was 120 h, the yield of fungal protein tended to be stable, reaching 8.93 g / L, and the productivity was 0.89 g / L / 12 h.

[0086] 2. Effects of different low-frequency ultrasound conditions on fungal protein production under single batch fermentation

[0087] Yellow pulp water containing 10% bean dregs was used as the culture medium and sterilized at 108°C for 20 min. After cooling, the spore suspension (10 6 spores / mL) were inoculated into yellow pulp water containing 10% bean dregs at a rate of 1% v / v for ultrasonic treatment. The ultrasonic conditions were as follows: fixed frequency of 23 kHz, pulse mode of 5 s on and 5 s off, ultrasonic density of 0-165.53 W / L, ultrasonic time of 4-16 min, and ultrasonic interval of 6-24 h. Single batch fermentation was carried out at 35°C and 150 rpm, and the fungal protein yield in different fermentation broths was measured every 12 h.

[0088] (1) Effects of different ultrasonic densities (0 W / L, 72.16 W / L, 116.20 W / L and 165.53 W / L) on fungal protein production. Figure 5 As shown in the figure, when the ultrasonic density was 116.20 W / L, the yield of fungal protein reached the maximum at 120 h, which was 11.18 g / L.

[0089] (2) Effects of different ultrasonic times (4 min, 8 min, 12 min and 16 min) on fungal protein production. Figure 6 As shown in the figure, when the ultrasonic time was 8 min, the yield of fungal protein reached the maximum at 108 h, which was 11.57 g / L.

[0090] (3) Effects of different ultrasonic intervals (6 h, 12 h, 18 h and 24 h) on fungal protein production. Figure 7 As shown in the figure, when the ultrasonic interval was 12 h, the yield of fungal protein reached the maximum at 96 h, which was 12.33 g / L.

[0091] By comparing the changing trends of fungal protein production in yellow pulp water containing 10% bean dregs under different low-frequency ultrasound conditions, the results showed that low-frequency ultrasound can not only increase the fungal protein production of Neurospora interformis OK in yellow pulp water containing 10% bean dregs, but also shorten the production time.

[0092] The repeated batch culture mode refers to replacing the culture medium for inoculating microorganisms with fresh culture medium at the end of batch fermentation. This technology is considered to be an effective means to improve product productivity. It not only shortens the culture time and eliminates the inoculation preparation step, but also avoids the time for cleaning and re-sterilization of the bioreactor, simplifying the operation process compared with traditional continuous culture. Therefore, based on the above optimal ultrasonic conditions, we adopted a repeated batch culture strategy to produce fungal protein.

[0093] 3. Effects of medium replacement ratio and number of replacements on the yield and productivity of fungal protein in repeated batch fermentation

[0094] The yellow pulp water containing 10% bean dregs was used as the fermentation medium and sterilized at 108℃ for 20 min. After cooling, the spore suspension (10 6 spores / mL) were inoculated into yellow pulp water containing 10% bean dregs at a rate of 1% v / v and ultrasonically treated. The ultrasonic conditions were as follows: fixed frequency of 23 kHz, pulse mode of 5 s on and 5 s off, ultrasonic density of 116.20 W / L, ultrasonic time of 8 min, and ultrasonic interval of 12 h. Repeated batch fermentation was carried out at 35°C and 120 rpm. When the reducing sugar concentration was lower than 0.5 g / L, 10-70% of the fermentation liquid was discharged, and an equal amount of fresh fermentation medium (yellow pulp water containing 10% bean dregs) was added. The culture was continued. When the reducing sugar concentration was lower than 0.5 g / L, 10-70% v / v of the fermentation liquid was discharged, and an equal amount of fresh fermentation medium was added. The above steps were repeated. When the yield of fermented fungal protein was lower than 50% of the yield of the previous batch of fermentation, the fermentation reaction was terminated. During this period, the yield of fungal protein in different fermentation liquids was measured every 12 h.

[0095] The effects of the replacement ratio and number of replacements of the fermentation medium on the yield and productivity of fungal protein in yellow slurry containing 10% bean dregs during repeated batch fermentation were compared. The results are shown in Table 5. When the number of replacements of the fermentation medium was 1 and the replacement ratio was 50%, the yield and productivity of fungal protein were the highest, which were 12.56 g / L and 1.71 g / L / 12h, respectively. At a replacement ratio of 50%, the number of replacements of the fermentation medium was compared. When the number of replacements of the fermentation medium was 3 times, the yield and productivity of fungal protein remained stable, and they were 12.20 g / L and 1.79 g / L / 12 h, respectively. Compared with Inter-type Neurospora OK, which was not treated with ultrasonic treatment and repeated batch culture, its yield and productivity in yellow slurry containing 10% bean dregs increased by 24.87% and 82.65%, respectively.

[0096] The above results show that low-frequency ultrasonic treatment and repeated batch culture can effectively increase the yield of fungal protein of Neurospora OK in yellow slurry water containing soybean dregs.

[0097] Table 5 Effects of different substitution ratios and substitution times on the yield and productivity of fungal protein

[0098]

[0099] Example 4 Changes in Chemical Oxygen Demand and Biological Oxygen Demand of Yellow Slurry Water for Fungal Protein Production

[0100] This example uses the determination method of chemical oxygen demand (COD) and the determination method of biological oxygen demand (BOD) to analyze the effect of ultrasonic treatment combined with repeated batch culture (as described in Example 3) on the removal rate of COD and BOD in yellow pulp water when 10% bean dregs are added.

[0101] Among them, the initial COD and BOD values ​​of yellow slurry water were 29038.67 mg / L and 9803.44 mg / L, respectively.

[0102] Among them, the determination method of the chemical oxygen demand (COD) refers to "GB / T 15456-2019 Determination of Chemical Oxygen Demand (COD) in Industrial Circulating Cooling Water - Permanganate Index Method"; the determination method of the biological oxygen demand (BOD) refers to "HJ505-2009 Dilution and Inoculation Method for Determination of Five-Day Biochemical Oxygen Demand (BOD5) of Water Quality".

[0103] The results showed that when 10% bean dregs were added and ultrasonic treatment combined with repeated batch culture strategy was used, the COD and BOD values ​​in yellow slurry water were reduced to 7720.03 mg / L and 550.95 mg / L, respectively, and the removal rates of the two reached 73.41% and 94.38%, respectively, which can reduce the burden of yellow slurry water as waste on the environment.

[0104] Example 5 Amino Acid Composition of Neurospora intermedius OK Fungal Protein

[0105] With reference to the method of "GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food", the crude protein content of the Neurospora intermedia fungal protein obtained from the yellow pulp water containing 10% bean dregs in Example 3 was determined by using ultrasonic treatment combined with repeated batch culture strategy. The crude protein content was 46.48%, which was slightly higher than the crude protein content of soybeans (44.6%), and much higher than the fungal protein of large fungi such as Pleurotus ostreatus, Agrocybe chaxini and Hericium erinaceus (25.3%~27.1%).

[0106] Referring to the method of GB 5009.124-2016 National Food Safety Standard Determination of Amino Acids in Food, the amino acid composition of Neurospora OK fungal protein was determined, and the results are shown in Table 6. Fungal protein contains 8 essential amino acids and 10 non-essential amino acids. The essential amino acid content of fungal protein is 185.48 mg / g, which exceeds the essential amino acid content of soybeans (174.03 mg / g). The ratios of essential amino acids / total amino acids and essential amino acids / non-essential amino acids of fungal protein are 42.75% and 74.71%, respectively, which meet the requirements of FAO / WHO for high protein (the ratios of essential amino acids / total amino acids and essential amino acids / non-essential amino acids of high-quality protein should be around 40% and above 60%, respectively).

[0107] Table 6 Amino acid composition of Neurospora intermedius OK fungal protein

[0108]

[0109] Example 6 Neurospora intermedia OK fungal protein used in the preparation of fried chicken nuggets

[0110] Collect the mycelium in the OK fermentation liquid of Neurospora intermedia, wash it, dry it, and grind it into powder to obtain fungal protein. Thaw the chicken breast at 4℃. Remove the fat and fascia on the surface of the thawed chicken, cut it into even-sized pieces, put it into a meat grinder and stir it for 2 minutes until the chicken is in a uniform state. Mix the chicken puree with the corresponding proportion of fungal protein, ice water, salt, sugar, pepper, and starch. The ingredients are shown in Table 7. Keep the temperature below 12℃ during the entire mixing process. After mixing, beat it quickly for 2 minutes, then add egg white and beat it for 2 minutes, and finally add corn oil and continue to beat it for 2 minutes, and put it in a -20℃ refrigerator for 1 hour. Take out the flour coating so that the surface of the chicken pieces is covered with a uniform layer of starch about 3mm thick. Fry the chicken pieces after flour coating at 120℃ for 4 minutes. Take them out after frying, raise the oil temperature to 180℃ and fry them quickly for 30 seconds.

[0111] Table 7 Chicken nugget ingredients and proportions replaced by different fungal proteins

[0112]

[0113] Note: The replacement ratio of fungal protein is calculated based on the dry weight of chicken breast.

[0114] (1) Determination of chicken nugget texture: The chicken nugget samples were cut into 1 cm × 1 cm × 1.5 cm cuboids and analyzed using a texture analyzer. The parameters were set as follows: P / 45 probe, measuring speed 1 mm / s, deformation 40%, trigger type automatic, trigger force 0.15 N, and time interval between two compressions 5 s.

[0115] Table 8 Chicken nugget texture analysis

[0116]

[0117] Note: Lowercase letters a, b, c indicate significant differences between different chicken nuggets with the same texture index ( p <0.05).

[0118] As shown in Table 8, as the proportion of mycoprotein replacing chicken breast in fried chicken nuggets increases, the hardness, adhesiveness, chewiness and shear force of fried chicken nuggets show a downward trend. The elasticity of chicken nuggets is enhanced. This shows that adding Neurospora OK mycoprotein to chicken nuggets can reduce the hardness of chicken nuggets and improve the elasticity and tenderness of chicken nuggets.

[0119] (2) Sensory evaluation: The chicken nuggets were subjected to sensory evaluation according to the scoring criteria in Table 9.

[0120] Table 9 Sensory scoring criteria for chicken nuggets

[0121]

[0122] The quality of chicken nuggets was comprehensively evaluated by sensory evaluation, and the sensory scores of chicken nuggets prepared by adding different amounts of Neurospora OK fungal protein are shown in Table 10. Compared with chicken nuggets without Neurospora OK fungal protein, chicken nuggets with Neurospora OK fungal protein added were better in appearance, shape, color, chewiness, toughness, aroma and taste. Among them, the chicken nuggets with a fungal protein replacement of 20% scored the highest.

[0123] Table 10 Sensory scores of chicken nuggets replaced by different fungal proteins

[0124]

[0125] Example 7 Neurospora intermedia OK fungal protein used in the preparation of vegetarian meat patties

[0126] First, make a basic vegetarian patty (No. A0), the formula of which is: 250 g / kg soy protein powder, 17 g / kg salt, 120 g / kg soybean oil, 60 g / kg starch, 25 g / kg soy sauce, 8 g / kg xanthan gum, 2 g / kg beet red, 65% water (calculated based on the water content in the finished product). Mix the raw materials thoroughly for 3-6 minutes and place them at 25°C for 50-60 minutes for marinating. Process the marinated raw materials into round patties (10 mm thick and 40 mm in diameter), freeze them at -20°C for about 30 minutes, and then bake them at 150°C for 15 minutes to make basic vegetarian patties.

[0127] According to the basic formula and process flow of vegetarian meat patties, fungal protein vegetarian meat patties with the addition of Neurospora OK fungal protein of 30%, 50% and 70% of the soy protein powder were made and numbered (A30, A50, A70).

[0128] (1) Determination of the texture of different vegetarian patties: The samples were subjected to texture profile analysis using a 50 mm disc probe in the “secondary compression” mode. Measurement parameters: compression ratio 80%, measurement speed 1 mm / s, trigger force 0.15 N, interval time 5 s. Hardness and elasticity were analyzed, and each sample was measured three times in parallel.

[0129] Table 11 shows the texture properties of different vegetarian patties. Compared with sample A0, the elasticity of the vegetarian patties increased significantly after adding 20% ​​and 50% fungal protein ( p <0.05). When the addition amount of fungal protein was 50%, the hardness of the vegetarian meat patty was the lowest and the elasticity was the highest, which indicated that the vegetarian meat patty made with 50% fungal protein replacement had the best texture.

[0130] Table 11 Comparison of the texture of vegetarian patties with different mycoprotein substitutes

[0131]

[0132] Note: Lowercase letters a, b, c indicate significant differences between different tofu with the same texture index ( p <0.05).

[0133] (2) Sensory quality analysis: The four types of vegetarian patties were cut into regular shapes of the same size, and each vegetarian patty sample was randomly numbered to ensure the objectivity of the sensory quality analysis. The sensory quality analysis was conducted based on five indicators: color, aroma, flavor, mouthfeel, and overall acceptability (Table 12).

[0134] Table 12 Sensory scoring criteria for vegetarian meat patties with different fungal protein substitutes

[0135]

[0136] from Figure 8 From the sensory evaluation of the four types of vegetarian patties, A50 mycelium patties were superior to other vegetarian patties in terms of aroma, color, taste and overall acceptability. This shows that it is feasible to use Neurospora intermedia fungal protein to make vegetarian patties, and the sensory evaluation of vegetarian patties with a 50% replacement of fungal protein is the best.

[0137] The present invention provides a strain of Neurospora intermedia and its application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A strain of Neurospora intermedia, characterized in that: The classification name is Neurospora intermedia, the strain number is OK, and it has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on April 10, 2024, with the deposit number CGMCC No.41150; Wherein, the Neurospora intermedia is fermented with yellow pulp water containing soybean dregs to produce cellulase and / or fungal protein; Wherein, the Neurospora intermedia is a Neurospora intermedia that reduces the chemical oxygen demand and biological oxygen demand in yellow pulp water.

2. Use of Neurospora intermedia according to claim 1 in producing cellulase.

3. Use of Neurospora intermedia according to claim 1 in producing fungal protein.

4. A method for producing fungal protein using Neurospora intermedia according to claim 1, characterized in that: The suspension of Neurospora intermedia spores is inoculated into a fermentation medium for fermentation culture, and then washed and dried to obtain the fungal protein.

5. The method according to claim 4, characterized in that The spore suspension has a concentration of 10 4 ~10 7 spores / mL, and the inoculation amount in the fermentation medium is 0.5-3% v / v.

6. The method according to claim 4, characterized in that The fermentation medium is yellow pulp water containing 5-10 wt% of bean dregs.

7. The method according to claim 4, characterized in that The fermentation culture adopts low-frequency ultrasound combined with repeated batch culture, and the specific steps are as follows: (1) inoculating a spore suspension of Neurospora intermedia into a fermentation medium for fermentation, and when the reducing sugar content is lower than 0.5 g / L, discharging 10 to 70% v / v of the fermentation liquid and adding an equal amount of fresh fermentation medium; (2) continuing to culture until the reducing sugar content is less than 0.5 g / L, continuing to discharge 10-70% v / v of the fermentation liquid and replenishing an equal amount of fresh fermentation medium; (3) Repeat step (2) and terminate the fermentation reaction when the yield of the fermented fungal protein is less than 50% of the yield of the previous batch of fermentation.

8. The method according to claim 7, characterized in that The fermentation culture conditions are: 24-37° C., 120-180 rpm; ultrasonic treatment is performed during the fermentation, and the ultrasonic conditions are: fixed frequency of 20-40 kHz, pulse mode of 3-6s on and 4-10s off, ultrasonic density of 0-165.53 W / L, ultrasonic time of 4-16 min, and ultrasonic interval of 6-24 h.

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