Fungus protein composition with high nutritional value and application thereof

Abstract

The invention discloses a fungal protein composition with high nutritional value and application thereof, and belongs to the technical field of nutritional evaluation of fungal proteins. The aspergillus niger mycelium protein structure is mainly flexible, and functional characteristics can be developed through conformation adjustment in the processing process. The aspergillus niger mycelium protein disclosed by the invention is good in foamability and has application advantages in foaming foods. And the protein can be easily absorbed by a human body, and is suitable for people with weak digestive functions or enteral nutrition preparation development.

Description

Technical Field

[0001] This invention relates to a fungal protein composition with high nutritional value and its application, belonging to the field of fungal protein nutritional evaluation technology. Background Technology

[0002] Protein, an essential macronutrient for the human body, plays a variety of physiological functions, including catalysis, transport, and immunity. Its basic building blocks are twenty amino acids, nine of which are essential amino acids that the human body cannot synthesize and must be obtained through diet to avoid nutritional deficiencies. For a long time, animal-derived proteins have been favored by consumers due to their rich content of the complete essential amino acid (EAA) profile. However, the sustainable development of modern animal husbandry faces severe challenges: the farming process not only consumes large amounts of water and land resources but also generates significant greenhouse gas emissions. In many scenarios, livestock farming has become a major source of terrestrial ecological pollution, directly leading to problems such as desertification and phosphorus cycle imbalances, while also releasing pollutants such as organic matter, pathogens, and drug residues into water bodies. Furthermore, excessive consumption of animal-based foods can lead to obesity, cardiovascular disease, metabolic syndrome, and gastrointestinal tumors due to increased saturated fat intake and decreased dietary fiber intake. Traditional animal husbandry can no longer sustainably meet global protein demands.

[0003] Against this backdrop, finding new alternative protein resources has become a crucial issue for the food industry. Fungal protein, as a highly promising meat protein substitute, is produced by cultivating microorganisms in fermenters and using renewable biomass as a substrate. Its unique advantages include: high nutritional value, containing all essential amino acids required by the human body; and significantly higher production efficiency (protein synthesis efficiency can be thousands of times higher than traditional animal husbandry), higher resource utilization, and lower carbon dioxide emissions compared to traditional animal husbandry. With continuous advancements in preparation technology, fungal protein is gradually becoming a safe and healthy alternative protein source, providing a feasible technological solution for human nutrition.

[0004] As a commonly used fermentation strain, *Aspergillus niger* has been gradually domesticated and improved into a multi-purpose cell factory capable of converting various inexpensive substrates into a series of valuable metabolites. However, current utilization of *Aspergillus niger* is mainly concentrated in the production of organic acids and protease preparations, with limited research on its mycelial proteins. Furthermore, existing *Aspergillus niger* mycelial proteins are primarily used in feed production, with limited applications in food processing. Moreover, the use of existing *Aspergillus niger* mycelial proteins in food processing faces several challenges. For example, many *Aspergillus niger* strains possess inherent toxicity, rendering their mycelial proteins unsuitable for food processing. Additionally, the molecular structures of some *Aspergillus niger* mycelial proteins are not well-suited for the preparation of protein products. Summary of the Invention

[0005] To address the above problems, the present invention provides a fungal protein composition with high nutritional value.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The first objective of this invention is to provide a fungal protein composition with high nutritional value, wherein the fungal protein composition is obtained by fermenting Aspergillus niger to obtain Aspergillus niger mycelium, and further extracting Aspergillus niger mycelial protein from the Aspergillus niger mycelium, wherein the preservation number of Aspergillus niger is CCTCC NO:M 2013703.

[0008] In one embodiment of the present invention, the fermentation of Aspergillus niger is carried out in a corn steep liquor culture medium, wherein the corn steep liquor culture is obtained by diluting the corn steep liquor solids content to 5% to 20% and then sterilizing it.

[0009] In one embodiment of the present invention, the specific steps of fermenting Aspergillus niger include:

[0010] S1. Dilute the corn steep liquor to a solids content of 5-20%, and sterilize it to obtain corn steep liquor culture medium;

[0011] S2. Inoculate 5-20% Aspergillus niger seed liquid into corn steep liquor medium according to the volume ratio, and ferment at 20-40℃ for 1-5 days to obtain Aspergillus niger mycelial fermentation broth.

[0012] In one embodiment of the present invention, the Aspergillus niger seed solution is obtained by inoculating a suspension of Aspergillus niger spores of not less than 1×106 CFU / mL into PDB medium at an inoculation rate of 1% to 3% and culturing at 28 to 32°C for 20 to 30 hours.

[0013] In one embodiment of the present invention, the Aspergillus niger spore suspension is obtained by eluting spores with physiological saline containing Tween-80 and then diluting.

[0014] In one embodiment of the present invention, in step S2, the rotation speed during fermentation is 400-600 r / min.

[0015] In one embodiment of the present invention, in step S2, the aeration ratio during fermentation is 0.4 to 0.6 vvm / L.

[0016] In one embodiment of the present invention, the extraction step specifically includes:

[0017] After breaking up the Aspergillus niger mycelium, adjust the pH to 9–11, sonicate at 240–260 W for 5–15 min, and then bathe in a water bath at 40–50 °C for 0.5–2 h. Centrifuge the extract at 6000–10000 r / min for 5–15 min, collect the supernatant, adjust the pH to 4–6, and centrifuge again at 6000–10000 r / min for 5–15 min to obtain mycelial protein.

[0018] A second objective of the present invention is to provide the application of the fungal protein composition in food processing.

[0019] In one embodiment of the present invention, the application is to prepare the fungal protein composition into an enteral nutrition preparation.

[0020] A third objective of this invention is to provide a method for evaluating fungal mycelial proteins, comprising the following steps:

[0021] Structural characteristics of fungal mycelial proteins were analyzed;

[0022] The functional characteristics of fungal mycelial proteins were analyzed.

[0023] Determine the in vitro digestibility and hydrolysis rate of fungal mycelial proteins;

[0024] The nutritional value and functional properties of fungal mycelial proteins were evaluated by comprehensively analyzing their structure, functional characteristics, and in vitro digestibility.

[0025] This invention analyzes the secondary structure of fungal mycelial proteins. Based on Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy data, it initially analyzes the structural characteristics of mycelial proteins from two fungal species. Further analysis of the tertiary structure of these proteins further distinguishes the compositional differences. This invention comprehensively utilizes structural and functional characterization analysis and in vitro digestion simulation technology to construct a systematic evaluation system for the nutritional value of fungal mycelial proteins. The research findings can provide theoretical support for the identification and evaluation of fungal germplasm resources, the comprehensive development of protein resources, and their high-value-added utilization. It helps clarify the core position of fungi in the discovery of novel protein resources and the development of protein supplement products, promoting the structural upgrading of the fungal industry from primary agricultural products to high-value-added, intensively processed products.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention provides a high-nutritional-value fungal protein composition. The Aspergillus niger mycelial protein of this invention has a predominantly flexible structure, which facilitates the expression of functional properties through conformational adjustments during processing. The Aspergillus niger mycelial protein of this invention exhibits good foaming properties, giving it an advantage in foamed foods. Furthermore, the protein of this invention is easily absorbed by the human body, making it suitable for individuals with weak digestive function or for the development of enteral nutrition formulations. Attached Figure Description

[0028] Figure 1 The figures show the infrared spectra of mycelial proteins from two fungi, with ab representing the infrared spectra of mycelial proteins from Aspergillus niger and Fusarium oxysporum, respectively.

[0029] Figure 2 The circular dichroism spectra of mycelial proteins from two fungi are shown.

[0030] Figure 3 The figures show the UV absorption spectra of mycelial proteins from two fungi, with ab representing the UV absorption spectra of Aspergillus niger mycelial protein and Fusarium mycelial protein, respectively.

[0031] Figure 4 The figures show the fluorescence spectra of mycelial proteins from two fungi, with ab representing the fluorescence spectra of mycelial proteins from Aspergillus niger and Fusarium oxysporum, respectively.

[0032] Figure 5 The functional properties of mycelial proteins from two fungi are analyzed. Figures a to c show the water-holding and oil-holding properties, foaming properties and stability, and emulsifying and stability of Aspergillus niger mycelial proteins and Fusarium mycelial proteins, respectively.

[0033] Figure 6 This image shows the in vitro simulated digestion analysis of mycelial proteins from two fungi. Figures ab and b represent gel electrophoresis images of Fusarium mycelial proteins and Aspergillus niger mycelial proteins, respectively. (M: 6 μL marker; 3–8 (a–f): gastric digestion stage (0, 5, 15, 30, 60, 120 min); 9–14 (g–l): intestinal digestion stage (0, 5, 15, 30, 60, 120 min); 1 (m): undigested group (TG enzyme + water); 2 (n): blank control (water is the test sample)).

[0034] Figure 7 The hydrolysis rate analysis of mycelial proteins of two fungi is shown in the figure. Figure ab shows the hydrolysis rate of Fusarium mycelial proteins and Aspergillus niger mycelial proteins, respectively.

[0035] Figure 8 The digestibility analysis of mycelial proteins from two fungi is shown in the figure. Figures ab and b represent the digestibility of Fusarium mycelial proteins and Aspergillus niger mycelial proteins, respectively. Detailed Implementation

[0036] This invention provides a method for comprehensively evaluating the nutritional and functional properties of fungal mycelial proteins, preferably comprising the following steps:

[0037] The structural characteristics of the fungal mycelial proteins were analyzed;

[0038] The functional characteristics of the fungal mycelial proteins were analyzed.

[0039] Determine the in vitro digestibility and hydrolysis rate of fungal mycelial proteins;

[0040] The nutritional value and functional properties of fungal mycelial proteins were evaluated by comprehensively analyzing their structure, functional characteristics, and in vitro digestibility.

[0041] This invention analyzes the secondary structure of mycelial proteins from two fungi, and preliminarily analyzes the structural characteristics of the mycelial proteins from two fungi based on Fourier transform infrared spectroscopy and circular dichroism spectroscopy data. Then, it further distinguishes the compositional differences of fungal mycelial proteins by analyzing the tertiary structure of the mycelial proteins.

[0042] The experimental materials are as follows:

[0043] Fusarium mycelial protein was obtained by fermentation of strains purchased from a strain bank. The samples were freeze-dried and stored in a cool, dry place for later use. Aspergillus niger (CCTCC NO: M 2013703) is disclosed in CN103937681A as a food-grade strain of Aspergillus niger and its application in the degradation of zearalenone.

[0044] PDA culture medium was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0047] Example 1:

[0048] Aspergillus niger CCTCC NO:M 2013703 was removed from -80℃, revived, and inoculated onto PDA medium. After culturing at 28℃ for 5 days, the spores were washed off with sterile physiological saline containing Tween-80, and the spore concentration was adjusted to 1×10⁻⁶. 6CFU / mL yields a suspension of Aspergillus niger spores. PDB medium is pumped into a 5L fermenter for sterilization. After sterilization and cooling, the temperature is raised to 30℃ and kept at that temperature. At a rotation speed of 500r / min, 2% Aspergillus niger spore suspension is added, and the culture time is 24h to obtain the seed culture medium.

[0049] Dilute corn steep liquor with a solid content of 40% to 10% and put it into a 10L liquid fermentation tank. The amount of liquid fermentation tank filled to 60% of the tank is 6L. Place the filled liquid fermentation tank into a high temperature and high pressure sterilizer at 121℃ for 20 minutes for sterilization. After sterilization, cool it down to below 30℃ and start inoculation.

[0050] Aspergillus niger seed culture was inoculated into corn steep liquor medium at an inoculation rate of 10%. Fermentation time was controlled at 60 h, with a controlled fermentation temperature of 25 °C, a rotation speed of 500 rpm, and an aeration ratio of 0.5 vvm / L. After fermentation, the mycelium was harvested through gauze and washed with water. The washed mycelium was then broken up, and the pH of the solution was adjusted to 10. The solution was then sonicated at 250 W for 10 min and incubated in a 45 °C water bath for 1 h. The extract was centrifuged at 8000 rpm for 10 min, the supernatant was collected, the pH was adjusted to 5, and the solution was centrifuged again at 8000 rpm for 10 min. The resulting precipitate was Aspergillus niger mycelial protein.

[0051] Test Example 1: Determination of the structural characteristics of mycelial proteins

[0052] The specific steps are as follows:

[0053] Place an appropriate amount of protein sample powder under the probe and record the infrared spectrum of the sample. Before measurement, perform an air background scan to calibrate the instrument and automatically subtract background noise. The spectral scanning range is 400–4000 cm⁻¹. -1 .

[0054] The secondary structure of the sample was measured using circular dichroism spectroscopy (CD). The protein was tested in the sample at a final concentration of 0.5 mg / mL under the following conditions: 25 °C, scanning wavelength 190–250 nm, 1.0 mm cuvette, scanning speed 60 nm / min, and optical path length 1.0 mm.

[0055] The tertiary structure of the samples was measured using a UV spectrophotometer. The protein concentration was 0.5 mg / mL, and the UV absorption spectra of the proteins were recorded in the wavelength range of 240 nm to 350 nm.

[0056] The tertiary structure of the samples was measured using a fluorescence spectrophotometer. The fluorescence spectra of the proteins were recorded in the emission wavelength range of 290 nm to 500 nm at an excitation wavelength of 280 nm.

[0057] Test Example 2: Determination of Functional Properties of Mycelial Proteins

[0058] The specific steps are as follows:

[0059] Disperse 50 mg of sample in a 15 mL centrifuge tube containing 5 mL of distilled water / corn oil. Weigh the sample and centrifuge tube (m1), stir magnetically for 30 min, let stand for a few minutes, then centrifuge at 20 °C and 4000 r / min for 15 min, and determine the mass of the precipitate and centrifuge tube (m2). Calculate the water holding capacity (WHC) and oil holding capacity (OHC) according to formulas (1) and (2):

[0060]

[0061] A protein solution with a concentration of 10 mg / mL was prepared by dispersing 50 mg of sample in a 50 mL centrifuge tube containing 5 mL of distilled water. This solution was then sheared at a high shear rate of 10000 r / min for 2 min. The foam volume at this point was measured, and its foaming property (FC) was calculated according to formula (3). After standing for 30 min, the foam volume was measured again, and its foam stability (FS) was calculated according to formula (4).

[0062]

[0063] Where: V is the initial solution volume (mL); V0 is the foam volume at 0 min after homogenization (mL); V 30 The volume of foam (mL) after standing for 30 minutes.

[0064] Take 40 mg of protein sample and put it into a centrifuge tube to prepare a protein solution with a concentration of 10 mg / mL. Homogenize the solution for 1 min on a high-speed homogenizer at a speed of 10000 r / min. Then add one-third of the liquid volume of vegetable oil and record the total volume of the solution (V0). Homogenize again and centrifuge the mixture at 12000 r / min for 10 min. Record the volume of the emulsion layer (V1). Then adjust the constant temperature water bath to 70℃ and put the sample after the first centrifugation in it. After 30 min, take it out and record the volume of the emulsion layer (V2). The emulsibility (EB) and emulsion stability (ES) are shown in Equations (5) and (6), respectively.

[0065]

[0066]

[0067] Test Example 3: Evaluation of Mycelial Protein Digestibility

[0068] The specific steps are as follows:

[0069] (1) Preparation of test samples

[0070] Before the simulated digestion, the protein concentration of each sample was adjusted to 10 mg / mL with deionized water (except for the blank control). The test sample groups are as follows.

[0071] Blank control: Water was used as the test sample, and acids, bases, electrolytes, enzymes, bile acids, etc. were added according to the simulated digestion process;

[0072] ANP (Aspergillus niger mycelial protein) digestion group: ANP solution was used as the test sample, and acids, bases, electrolytes, enzymes, bile acids, etc. were added according to the simulated digestion process;

[0073] ANP undigested group: ANP solution was used as the test sample and diluted with deionized water to the same volume as the simulated final digestion volume.

[0074] FP (Fusarium mycelium protein) digestion group: FP solution was used as the test sample, and acids, bases, electrolytes, enzymes, bile acids, etc. were added according to the simulated digestion process;

[0075] FP undigested group: FP solution was used as the test sample and diluted with deionized water to the same volume as the simulated final digestion volume;

[0076] (2) Stomach digestion stage

[0077] Take a 50 mL centrifuge tube and add 5 mL of the test sample. Preheat the gastric electrolyte at 37 °C and add 4 mL of 1.25 × gastric digestion electrolyte. Add 5 mol / L HCl to adjust the pH to 3.0. Add porcine pepsin solution prepared in 0.1 mmol / L HCl to achieve a pepsin activity of 2000 U / mL in the final digestion mixture. Confirm the system pH is 3. Add deionized water to make the total volume 10 mL. Shake at 37 °C for 2 h and take samples at 0, 5, 15, 30, 60, and 120 min. Add 5 mol / L NaOH to adjust the pH to 7 to terminate the gastric digestion stage.

[0078] (3) Intestinal digestion stage

[0079] Preheat the intestinal fluid electrolyte at 37℃. Take 5 mL of the solution after gastric digestion and add 3 mL of 1.25× intestinal fluid digestion electrolyte. Add bovine bile solution prepared in deionized water to achieve a bile acid content of 10 mmol / L in the digestion mixture. Incubate at 37℃ and vortex for 30 min to completely dissolve the bile. Add porcine pancreatin prepared in deionized water to achieve a trypsin activity of 100 U / mL in the final mixture. Confirm the pH of the system is 7. Add deionized water to make a total volume of 10 mL. Shake at 37℃ for 2 h and take samples at 0, 5, 15, 30, 60, and 120 min.

[0080] (4) Termination of digestion

[0081] Add 300mM trypsin inhibitor solution, bringing the final inhibitor concentration to 30mM. Shake to mix, centrifuge at 4000r / min for 20min, collect the supernatant, and store at -80℃.

[0082] (5) SDS-PAGE gel electrophoresis

[0083] The simulated digested test samples were mixed with SDS-PAGE protein loading buffer (5×) at a ratio of 4:1 (v / v), heated to 100℃ in a metal bath for 4 min to obtain the corresponding protein loading solutions for each group of samples. Protein electrophoresis gels were prepared using a one-step PAGE gel rapid preparation kit (12.5%) according to the manufacturer's instructions. The prepared gel plates were inserted into the electrophoresis apparatus, and electrophoresis buffer (1×) was poured in until the gel plates were completely submerged. 20 μL of the protein loading solution for each group was added to each lane, and 6 μL of pre-stained protein molecular weight standards (10-180 kD) was added to another lane. Electrophoresis was performed at a constant voltage of 120 V for approximately 2 h, stopping when the bromophenol blue indicator reached the lower edge of the separating gel. The gels were removed, rinsed three times with water, and stained and washed according to the instructions for Coomassie Brilliant Blue rapid staining solution. Gel images were taken using a gel imaging system. The digested samples were diluted with PBS to 2.5 mg / mL, centrifuged, and then the protein molecular weight distribution was determined.

[0084] (6) Protein hydrolysis rate determination

[0085] The degree of hydrolysis (DH) after digestion was determined by the o-phthalaldehyde (OPA) assay. The protein digest was mixed with 10% cooled trichloroacetic acid (TCA, w / v) at a 1:1 volume ratio, allowed to stand for 2 h, and then centrifuged at 10,000 × g for 15 min. 10 μL of the supernatant was then added to a 96-well transparent polystyrene plate, along with 200 μL of OPA working reagent (o-phthalaldehyde (OPA) (0.8 mg / mL), dithiothreitol (DTT) (0.1 w / v), and borate buffer (pH 9.3)). A standard curve was prepared using 0–1 mg / mL L-leucine. The 96-well plate was incubated in the dark for 3 min, and the absorbance was measured at 340 nm using a microplate reader. DH was calculated using the following formula:

[0086]

[0087] NH2(final) refers to the concentration of free amino groups at different sampling points, NH2(initial) refers to the concentration of free amino groups before digestion, and NH2(acid) refers to the concentration of total free amino groups after acid hydrolysis (6M HCl, 110℃, 24h).

[0088] (7) Protein digestibility determination

[0089] Mix 0.1 mL of the digested gastric or intestinal reaction solution with 0.1 mL of 10% TCA and incubate at 4°C for 2 h to obtain undigested protein. Centrifuge the mixture at 10,000 × g for 15 min. Resuspend the precipitate in 0.5 mL of PBS and determine the protein content using a Bradford protein assay kit. Repeat each sample three times. Protein digestibility (%) is calculated using the following formula:

[0090]

[0091] Where W0 and W1 represent the protein content of the samples before and after digestion.

[0092] Results Analysis

[0093] 1. Structural characteristic evaluation

[0094] 1.1 Infrared Spectroscopic Analysis

[0095] The backbone atoms of a polypeptide chain coil or fold along certain axes to form a specific conformation. This spatial arrangement of the backbone atoms contains rich information about the protein's secondary structure, and therefore, FTIR can be used to reflect the protein's conformation and the stretching vibrations of functional groups. The secondary structures of ANP and FP were detected and analyzed; the infrared spectra are shown below. Figure 1 Both proteins are at 3300 cm⁻¹ -1The presence of distinct absorption peaks on both sides is caused by the NH stretching vibration of the primary amine. (2800 cm⁻¹) -1 ~3000cm -1 The increased absorbance of the absorption peaks appearing in the band is related to the symmetric and asymmetric stretching vibrations of the methyl and methylene groups in proteins; 1600 cm⁻¹ -1 ~1700cm -1 and 1500cm -1 ~1600cm -1 The characteristic absorption peaks appearing in the bands are located in the amide I and amide II bands, respectively, which are typical features of protein secondary structure (β-sheet and α-helix). They are mainly caused by the stretching vibration of CO, the bending vibration of NH and the stretching vibration of CN in the protein peptide bond, confirming that both proteins have complete secondary structures.

[0096] 1.2 Circular Dichroism Spectroscopy Analysis

[0097] Table 1. Secondary structure content of protein samples

[0098]

[0099] CD spectroscopy analyzes the secondary structure of proteins by measuring the difference in absorption of left-handed and right-handed circularly polarized light. Different secondary structures exhibit characteristic absorption peaks at specific wavelengths. The secondary structures of two proteins were detected using CD spectroscopy; the results are shown below. Figure 2 Negative peaks were observed in both proteins around 208 nm, indicating the presence of α-helical structures, while a weak positive peak appeared around 215 nm in the β-sheet structure. The proportions of secondary structures in the two proteins were calculated through fitting analysis of the circular dichroism spectral data. The table shows that the contents and proportions of various structures in Aspergillus niger mycelial proteins and Fusarium mycelial proteins are similar. The α-helix has the highest content, accounting for 56.8% and 56.0% respectively, indicating that both ANP and FP rely on helical structures to maintain catalytic core or transmembrane stability. β-sheets are formed by multiple β-chain segments laterally connected by hydrogen bonds to form a sheet structure. The hydrogen bond network gives this structure high stability, making it relatively rigid and resistant to deformation. However, the β-sheet content of ANP is only 4.4%, and that of FP is 4.0%, the lowest proportions, indicating fewer regularly arranged rigid regions in the structure, and that the protein is mainly composed of flexible structures.

[0100] 1.3 Ultraviolet Absorption Spectroscopy Analysis

[0101] like Figure 3As shown, the maximum UV absorption wavelength of ANP is 283 nm, and that of FP is 282 nm. Both proteins exhibit characteristic absorption around 280 nm, mainly due to the presence of aromatic amino acids (such as tryptophan, tyrosine, and phenylalanine). Furthermore, the highest UV absorption peak of FP appears at 1.8 nm at 282 nm, while that of ANP is 1.3 nm, indicating that the content of aromatic amino acids in FP is relatively higher than that in ANP.

[0102] 1.4 Fluorescence Spectroscopy Analysis

[0103] Tryptophan is often used as an endogenous fluorescent probe to detect changes in protein tertiary structure. Tryptophan-containing residues fluoresce in the 300nm–400nm range when excited at 290nm. Figure 4 It can be seen that ANP exhibits the highest tryptophan fluorescence intensity at 336 nm when excited at 290 nm, while FP exhibits the highest tryptophan fluorescence intensity at 331 nm when excited at 290 nm. Fluorescence intensity increases with increasing protein content, indicating increased external exposure of tryptophan residues. However, the fluorescence spectral peaks do not change with different protein concentrations. The difference lies in the fact that, at the same protein concentration, the peak value of FP is significantly higher than that of ANP. For example, at a protein concentration of 0.5 mg / ml, the fluorescence intensity of FP is 45000 AU, far exceeding that of ANP (37000 AU), indicating that the tryptophan content in FP is higher than that in ANP.

[0104] 2. Analysis of the functional characteristics of fungal mycelial proteins

[0105] like Figure 5 As shown, FP significantly outperforms ANP in terms of water retention. FP has a water-holding capacity of 0.045 g / g, while ANP has 0.02 g / g. This indicates that FP has a better ability to bind and retain moisture. This characteristic gives FP an advantage in applications requiring moisture retention, such as food processing, where it may be more beneficial for maintaining product moisture and softness, thus helping to extend shelf life. In terms of oil retention, both have similar oil-holding capacities, approximately 0.05 g / g, meaning they have similar abilities to bind and retain oils.

[0106] Protein foaming properties refer to the amphiphilic structure that allows molecules to orient themselves at the air-water interface, reducing surface tension and thus promoting bubble formation. ANP exhibits superior foaming properties, with a value of approximately 90%, while FP's value is approximately 70%. This high foaming property allows ANP to quickly form rich foam structures when mixed with other ingredients. FP's foaming stability is 55%, slightly higher than ANP's 50%. This indicates that the foam formed by FP can be maintained for a longer period.

[0107] FP's emulsifying capacity is 60%, which is better than ANP's (40%), indicating that FP has a better ability to uniformly mix two immiscible liquids to form a stable emulsion. In terms of emulsifying stability, ANP has a higher emulsifying stability of 80%, indicating that the emulsion formed by ANP can remain stable for a longer period of time.

[0108] 3. Effects of in vitro simulated digestion on mycelial proteins

[0109] 3.1 In vitro simulated digestion

[0110] Table 2. Distribution of protein molecular weight before and after digestion

[0111]

[0112] It is an important method for elucidating the mechanisms of protein hydrolysis. For example... Figure 6 As shown, the electrophoretic band intensities of Aspergillus niger mycelial protein (ANP) and Fusarium mycelial protein (FP) remained almost unchanged compared to their initial state during gastric digestion. However, the change in protein molecular weight distribution during digestion revealed a significant decrease in the content of high molecular weight proteins and an increasing trend in the content of low molecular weight proteins. This indicates that both proteins underwent digestion and degradation at this stage, although some high molecular weight proteins remained, suggesting that these two proteins exhibit significant resistance to gastric digestion. Upon entering the intestinal digestion stage, the intensity of the high molecular weight regions (>40 kDa) of ANP and FP protein bands significantly weakened or even disappeared completely, clearly demonstrating a gradual degradation process. This indicates that intestinal digestive enzymes have a strong degradative effect on these two proteins. Specifically, during intestinal digestion, ANP showed increased band intensity in the 30–37 kDa range as high molecular weight proteins were gradually degraded, indicating that the large molecular structure was hydrolyzed into smaller molecular weight proteins. The bands then disappeared as digestion progressed. Although FP did not show a clear band distribution, the band intensity gradually weakened, and the bands essentially disappeared by the end of intestinal digestion. The protein molecular weight distribution map after intestinal digestion shows that low molecular weight proteins account for the largest proportion (FP 48.93%, ANP 47.94%), further confirming the electrophoresis results. Based on these results, it can be inferred that ANP and FP have resistance to gastric digestion; after intestinal digestion, most proteins are hydrolyzed.

[0113] 3.2 Hydrolysis rate

[0114] Degree of hydrolysis (DH) is defined as the proportion of peptide bonds broken in protein hydrolysates. The OPA method is a commonly used method to characterize the degree of protein hydrolysis by quantifying the relative amount of free amino groups in the system. The changes in the degree of protein hydrolysis (FP and ANP) at different time points are shown below. Figure 7As shown in the figure, during the gastric digestion stage, the degree of hydrolysis of each group of samples increased only slightly. The free amino acid content of FP increased slightly after gastric digestion compared to the undigested sample, reaching 10% after 120 minutes of gastric digestion. In contrast, the degree of hydrolysis of ANP increased only slightly during gastric digestion, by about 2%. During the intestinal digestion stage, the free amino acid content of both FP and ANP increased significantly compared to the gastric digestion stage. After 5 minutes of intestinal digestion, the free amino acid content of FP surged to 75%, while the free amino acid content of the ANP sample reached 25%. Furthermore, the degree of hydrolysis of FP remained higher than that of ANP throughout the digestion process, indicating that under the action of trypsin, large protein molecules were broken down into smaller amino acids and a small amount of peptides.

[0115] 3.3 Digestibility

[0116] Depend on Figure 8 It was found that during the gastric digestion phase, the digestibility rates of FP and ANP were relatively low, reaching 6.8% and 3.6% respectively after gastric digestion. However, their digestibility rates increased significantly after the start of the intestinal digestion phase, and slowed considerably at 135 minutes. By the end of digestion, the digestibility of FP reached 70%, and that of ANP reached 60%. Throughout the simulated gastrointestinal digestion process, the digestibility of FP was higher than that of ANP, especially during the intestinal digestion phase (120–240 minutes). This indicates that FP is more easily digested and facilitates gastric emptying, making it more suitable for individuals with digestive and malabsorption disorders. It can improve tolerance to enteral nutrition and thus improve nutritional status.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fungal protein composition with high nutritional value, characterized in that, The fungal protein composition is obtained by fermenting Aspergillus niger to obtain Aspergillus niger mycelium, and then extracting Aspergillus niger mycelial protein from the Aspergillus niger mycelium. The preservation number of Aspergillus niger is CCTCC NO:M 2013703.

2. The fungal protein composition according to claim 1, characterized in that, The fermented Aspergillus niger is fermented in corn steep liquor culture medium, which is obtained by diluting the corn steep liquor solids content to 5% to 20% and then sterilizing it.

3. The fungal protein composition according to claim 2, characterized in that, The specific steps for fermenting Aspergillus niger include: S1. Dilute the corn steep liquor to a solids content of 5-20%, and sterilize it to obtain corn steep liquor culture medium; S2. Inoculate 5-20% Aspergillus niger seed liquid into corn steep liquor medium according to the volume ratio, and ferment at 20-40℃ for 1-5 days to obtain Aspergillus niger mycelial fermentation broth.

4. The fungal protein composition according to claim 3, characterized in that, The Aspergillus niger seed solution is obtained by inoculating a suspension of Aspergillus niger spores of not less than 1×106 CFU / mL into PDB medium at an inoculation rate of 1% to 3% and culturing at 28 to 32°C for 20 to 30 hours.

5. The fungal protein composition according to claim 4, characterized in that, The Aspergillus niger spore suspension was obtained by eluting spores with physiological saline containing Tween-80 and then diluting.

6. The fungal protein composition according to claim 3, characterized in that, In step S2, the fermentation speed is 400–600 r / min and the aeration ratio is 0.4–0.6 vvm / L.

7. The fungal protein composition according to claim 1, characterized in that, The extraction steps specifically include: After breaking up the Aspergillus niger mycelium, adjust the pH to 9–11, sonicate at 240–260 W for 5–15 min, and then bathe in a water bath at 40–50 °C for 0.5–2 h. Centrifuge the extract at 6000–10000 r / min for 5–15 min, collect the supernatant, adjust the pH to 4–6, and centrifuge again at 6000–10000 r / min for 5–15 min to obtain mycelial protein.

8. The use of the fungal protein composition according to any one of claims 1 to 7 in food processing.

9. The application according to claim 8, characterized in that, The application involves preparing the fungal protein composition into an enteral nutrition formulation.

10. A method for evaluating fungal mycelial proteins, characterized in that, Includes the following steps: Structural characteristics of fungal mycelial proteins were analyzed; The functional characteristics of fungal mycelial proteins were analyzed. Determine the in vitro digestibility and hydrolysis rate of fungal mycelial proteins; The nutritional value and functional properties of fungal mycelial proteins were evaluated by comprehensively analyzing their structure, functional characteristics, and in vitro digestibility.

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