Fusarium venenatum protein, low-lipid protein foodstuffs and methods and uses thereof

Abstract

The present application belongs to the field of food science and microbial engineering, and specifically relates to a fusarium venenatum protein, a low-fat protein food and a preparation method and use thereof. The present application provides a high-emulsifying fusarium venenatum TB01 protein, a preparation method and application thereof. The present application uses fusarium venenatum TB01 mycelium as raw material, and innovatively realizes the functional modification of protein structure by sequentially cooperating complex enzymolysis and ultrasonic treatment, thereby efficiently breaking the cell wall to extract high-content protein (≥82%) and realizing the functional modification of protein structure. Tests show that the emulsification activity and stability of the obtained protein are significantly better than those of soybean protein, and the emulsification stability is better than that of commercial whey protein. Therefore, the protein obtained by the present application can be used as a high-performance emulsifying ingredient to replace or partially replace whey protein or soybean protein, and is applied to beverage, plant yogurt, sauce and other food systems.

Description

Technical Field

[0001] This invention belongs to the field of food science and microbial engineering, specifically relating to Fusarium vesicatoria protein, low-fat protein foods, their preparation methods and uses. Background Technology

[0002] With global population growth and the demand for sustainable food systems, the search for novel and sustainable protein sources has become a research hotspot. Plant proteins (such as soy protein) and animal proteins (such as whey protein) are currently the mainstream functional protein raw materials, but they face challenges such as land resource consumption, allergens, or supply fluctuations.

[0003] Venetian Fusarium ( Fusarium venenatum As a promising source of protein from single-celled fungi, whey protein offers advantages such as rapid growth, wide availability of culture media, and rich nutrition. However, the tough cell wall structure of fungi often results in unsatisfactory protein yields and functionalities when directly extracted. In existing technologies, enzymatic hydrolysis is used for cell wall disruption, but its efficiency is limited. Ultrasonic treatment, as a physical method for cell wall disruption, faces a trade-off between energy consumption and the risk of protein denaturation. Currently, there are no reports of extracting high-purity protein products from Fusarium venetum strain TB01 that possess key emulsifying functions comparable to whey protein and soy protein through the synergistic effect of specific complex enzymes and optimized ultrasonic processes.

[0004] Therefore, it is of great significance to develop a process that can efficiently extract high-functionality proteins from fungal mycelia and obtain high-performance protein products. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a synergistic preparation method for efficiently extracting highly emulsifying isolated proteins from Fusarium tumefaciens TB01 mycelium. This method solves the problems of low cell wall breaking efficiency and insufficient protein functionality in existing technologies, thereby obtaining a fungal protein product that is comparable to whey protein and significantly superior to soybean protein in key emulsifying properties, and expanding its application value in high-end emulsified food systems.

[0006] This invention provides a Fusarium vesicatoria protein, which is prepared by comprising the following steps: Step 1: Mix Fusarium vesicatoria with cell wall lysin for enzymatic hydrolysis. Step 2: The enzymatic hydrolysis product is extracted with alkali to obtain an alkali extract with an alkali concentration of 40-60 mM. Step 3: Sonicate the alkaline extract at a power density of 300-400 W / L, then add acid to obtain the final product.

[0007] Furthermore, in step 1, the cell wall lysing enzyme is a complex enzyme composed of a breakdown enzyme, a fungal cell wall lysing enzyme, a yeast cell wall lysing enzyme, and a snail enzyme, with a mass ratio of 1-2:1-2:1-2:1-2.

[0008] Preferably, the cell wall lysing enzyme is a complex enzyme composed of a breakdown enzyme, a fungal cell wall lysing enzyme, a yeast cell wall lysing enzyme, and a snail enzyme, with a mass ratio of 1:1:2:1.

[0009] Furthermore, in step 1, the conditions for the enzymatic hydrolysis reaction include: a temperature of 37-40℃, a pH of 6-6.5, and a reaction time of 2-3 hours.

[0010] Furthermore, in step 1, before mixing Fusarium venetum with cell wall dissolving enzyme, it is pre-dissolved in water, and the mass ratio of Fusarium venetum dry powder to water is 1:40-60. And / or, in step 1, the amount of cell wall dissolving enzyme added is 2% or 4% to 6% of the dry basis.

[0011] Furthermore, in step 2, the alkali is an inorganic strong alkali, and the alkali extraction conditions include: extraction time of 0.5-2 hours and extraction temperature of 40-60℃.

[0012] Furthermore, in step 3, the ultrasound time is 5-30 minutes.

[0013] Furthermore, in step 3, the specific process of adding acid includes: adjusting the pH to 3.6-4.0 with acid, and letting it stand at 4-6 ℃ for 8-12 hours; And / or, in step 3, the acid is an inorganic strong acid; after the acid addition operation, centrifugation, washing, alkali neutralization, and drying are performed.

[0014] This invention provides a method for preparing Fusarium vesicatoria protein as described in any of the preceding claims, comprising the following steps: Step 1: Mix Fusarium vesicatoria with cell wall lysin for enzymatic hydrolysis. Step 2: The enzymatic hydrolysis product is extracted with alkali to obtain an alkali extract with an alkali concentration of 40-60 mM. Step 3: Sonicate the alkaline extract at a power density of 300-400 W / L, then add acid to obtain the final product.

[0015] The present invention provides the use of Fusarium vesicatoria protein as described in any of the preceding claims in the preparation of protein foods as an emulsifying ingredient.

[0016] The present invention provides a low-fat protein food comprising Fusarium vesicatoria protein as described in any of the preceding claims.

[0017] By screening the process conditions and operational sequence of enzymatic hydrolysis, alkali extraction, and ultrasonic treatment, this invention provides a highly emulsifying Fusarium venetum TB01 protein, its preparation method, and its applications. This invention uses Fusarium venetum TB01 mycelium as raw material and innovatively combines enzymatic hydrolysis and ultrasonic treatment in a synergistic sequence, achieving efficient cell wall disruption and extraction of high-content protein (≥82%) while simultaneously functionalizing the protein structure. Testing showed that the obtained protein exhibited significantly better emulsifying activity and stability than soy protein, and its emulsifying stability was superior to commercial whey protein. Therefore, the protein obtained by this invention can be used as a high-performance emulsifying ingredient to replace or partially replace whey protein or soy protein in food systems such as beverages, plant-based yogurt, and sauces.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 This is a diagram of the high internal phase lipid mimicry of protein FV1 of this invention; Figure 2 The figure shows the effect of different enzymatic hydrolysis schemes on protein extraction rate; among them, Figure 2 Figure A shows the screening results for the proportion of complex enzymes. Figure 2 B shows the screening results for enzymatic hydrolysis temperature. Figure 2 Figure C shows the screening results for enzymatic hydrolysis pH. Figure 2 Figure D shows the screening results for the amount of compound enzyme added. Figure 2 E is the screening result diagram of the solid-liquid ratio of mycelial dry powder to water. Figure 2 F represents the screening results of enzymatic hydrolysis time, where h represents hours; Figure 3 The graph shows the effect of different alkaline extraction methods on protein extraction rate; among them, Figure 3 Figure A shows the screening results for alkali extraction time. Figure 3 B shows the screening results for alkali extraction temperature. Figure 3 C represents the screening results for alkali concentration; Figure 4 The figure shows the effect of ultrasound-assisted extraction on protein extraction rate; among them, Figure 4 A shows the screening results for ultrasonic power density. Figure 4 B shows the screening results of ultrasound time, where min represents minutes; Figure 5 This is a visual comparison chart of protein FV1 of the present invention with commercially available soy protein and whey protein emulsions, where SP represents soy protein isolate, WP represents whey protein isolate, and FV1 is the protein prepared in Example 1 of the present invention. Figure 6 The graph shows the comparison of surface tension at the oil-water interface between protein FV1 of the present invention and commercially available soy protein and whey protein. In this graph, SP represents soy protein isolate, WP represents whey protein isolate, and FV1 is the protein prepared in Example 1 of the present invention. Figure 2-4 In this study, the significance of differences between groups was indicated by letter notation, with different lowercase letters indicating significant differences between groups (one-way ANOVA, Duncan's multiple comparison test, p<0.05). Detailed Implementation

[0021] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0022] The raw material Fusarium venetum TB01 used in the following examples was listed as a new food ingredient in Document No. 7 of the National Health Commission of the People's Republic of China in 2025 and was provided by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.

[0023] Example 1: A method for preparing highly emulsifiable Fusarium venetum TB01 protein This embodiment provides a method for preparing highly emulsifying Fusarium tumefaciens TB01 protein. This method employs optimal enzymatic hydrolysis, optimal alkali extraction parameters, and ultrasound-assisted extraction, specifically comprising the following steps: S1. Weigh 2.0 g of dried mycelial powder of Fusarium tumefaciens TB01, add 100 mL of ultrapure water (solid-liquid ratio 1:50), and hydrate with magnetic stirring at room temperature for 2 h.

[0024] S2. Adjust the pH of the mixture to 6.0. Add a compound enzyme (Driselase:Lywallzyme:Zymolyase:Snailase = 1:1:2:1), with a total addition of 4.0% of the dry basis. Incubate at 37 °C for 2 h.

[0025] Among them, the enzymes in the above compound enzymes are all cell wall dissolving enzymes produced by Shanghai Maokang Biotechnology Co., Ltd.: Driselase (dissolving enzyme, derived from Basidiomycetes, catalog number: MX7355-1G-T), Lywallzyme (fungal cell wall dissolving enzyme, derived from Trichoderma longifolia, catalog number: MX7365-1G), Zymolyase (yeast cell wall dissolving enzyme, derived from Arthrobacter flavus, catalog number: MF0212-1G), and Snailase (snail enzyme, catalog number: MF0213-5G).

[0026] S3. Add 0.15 M NaOH solution (volume ratio 2:1) to the above enzymatic hydrolysate to make the final NaOH concentration 50 mM. Perform alkaline extraction for 1.5 h with stirring at 40 ℃ and 800 rpm.

[0027] S4. Sonicate the alkaline extract at a power density of 300 W / L for 10 min (pulse mode, 1s on / 1s off), and control the temperature to <40 ℃.

[0028] S5. Adjust the pH of the solution to 3.8 with 1M HCl, let it stand at 4℃ for 12 h, centrifuge, wash, reconstitute, adjust the pH to 7.0, and then freeze-dry. Protein powder FV1 is obtained.

[0029] Example 2: A method for preparing highly emulsifiable Fusarium venetum TB01 protein The same preparation method as in Example 1 was used, except that in step S3, the final concentration of the alkali solution was adjusted to 30 mM.

[0030] In this embodiment, protein powder FV2 was prepared.

[0031] Example 3: A method for preparing highly emulsifiable Fusarium venetum TB01 protein The same preparation method as in Example 1 was used, except that in step S3, the final concentration of the alkali solution was adjusted to 60 mM.

[0032] In this embodiment, protein powder FV3 was prepared.

[0033] Example 4: Highly emulsifiable Fusarium vesicae TB01 protein used to prepare high internal phase fat mimicry This embodiment uses the protein prepared in Example 1 to prepare a high internal phase fat mimicry, which is a novel fat substitute for developing low-fat or zero-trans-fat foods.

[0034] The preparation method is as follows: the protein is prepared into a 4% wt solution, and then mixed with sunflower seed oil at a volume ratio of 1:3, and sheared at 12,000 rpm for 2 min.

[0035] The high-internal-phase fat mimicry prepared in this embodiment is as follows: Figure 1 As shown.

[0036] Comparative Example 1 The same preparation method as in Example 1 was used, except that step S4 was omitted, and the alkaline extract obtained after step S3 was directly subjected to acid precipitation, washing, and freeze-drying according to the method in step S5. Protein powder FV4 was obtained.

[0037] Comparative Example 2 C1: Same as step S1 in Example 1.

[0038] C2: After hydration, the mycelial liquid is not enzymatically hydrolyzed, but directly mixed with NaOH at a volume ratio of 2:1 to prepare a 50 mM alkaline solution, and extracted by stirring at 40℃ for 2 h.

[0039] C3: No ultrasonic treatment. The extract obtained in step C2 was adjusted to pH 3.8 for precipitation, allowed to stand at 4 ℃ for 12 h, centrifuged, washed, reconstituted, and adjusted to pH 7.0 before lyophilization. Protein powder FV5 was obtained.

[0040] The technical solution of the present invention will be further explained through experiments below. The protein samples FV1, FV2, FV3, FV4 and FV5 detected in the following experimental examples were prepared by the methods of the above examples or comparative examples.

[0041] Experiment Example 1: Screening Experiment for Enzymatic Digestion Conditions I. Experimental Methods 1. Screening experiment for the ratio of compound enzymes Enzymatic hydrolysates were prepared according to steps S1 and S2 of Example 1, wherein the ratio of the complex enzyme in step S2 was 1:1:1:1, 2:1:1:1, 1:2:1:1, 1:1:2:1, and 1:1:1:2, respectively. The protein extraction rate of each group was measured.

[0042] 2. Screening experiment for enzymatic hydrolysis temperature The enzymatic hydrolysate was prepared according to steps S1 and S2 of Example 1, wherein the enzymatic hydrolysis temperatures of the composite enzyme in step S2 were 30℃, 33℃, 35℃, 37℃, and 40℃, respectively. The protein extraction rate of each group was measured.

[0043] 3. Screening experiment for pH of enzymatic hydrolysis The enzymatic hydrolysate was prepared according to steps S1 and S2 of Example 1, wherein in step S2, the pH of the mixture was adjusted to 5, 5.5, 6, 6.5, and 7, respectively. The protein extraction rate of each group was measured.

[0044] 4. Screening experiment on the amount of compound enzyme added The enzymatic hydrolysate was prepared according to steps S1 and S2 of Example 1, wherein the total amount of the compound enzyme added in step S2 was 2%, 3%, 4%, 5%, and 6%, respectively. The protein extraction rate of each group was measured.

[0045] 5. Screening experiment for solid-liquid ratio The enzymatic hydrolysate was prepared according to steps S1 and S2 of Example 1, wherein in step S1, the solid-liquid ratio of mycelial powder to water was 1:20, 1:30, 1:40, 1:50, and 1:60, respectively, and the mass of the mycelial powder was the same as in Example 1. The protein extraction rate of each group was measured.

[0046] 6. Screening experiment for enzymatic hydrolysis time The enzymatic hydrolysate was prepared according to steps S1 and S2 of Example 1, wherein the enzymatic hydrolysis time in step S2 was 1 h, 1.5 h, 2 h, 2.5 h, and 3 h, respectively. The protein extraction rate of each group was measured.

[0047] 7. Determination of protein extraction rate Protein extraction rate = Mass of protein in the supernatant of the enzymatic hydrolysate / Total mass of protein in the raw material × 100%.

[0048] II. Experimental Results 1. Screening results of the ratio of compound enzymes The results are as follows Figure 2 As shown in Figure A, the results indicated that the protein extraction rate was significantly higher when the ratio of the complex enzyme (Driselase:Lywallzyme:Zymolyase:Snailase) was 1:1:2:1 than that of other groups. Furthermore, the protein extraction rates at ratios of 1:1:1:1, 2:1:1:1, and 1:2:1:1 were also significantly higher than that at 1:1:1:2. Therefore, the preferred ratio of the complex enzyme (Driselase:Lywallzyme:Zymolyase:Snailase) in this experimental example is 1:1:2:1.

[0049] 2. Screening results for enzymatic hydrolysis temperature The results are as follows Figure 2 As shown in Figure B, the results indicate that the enzymatic hydrolysis temperature has a significant impact on the protein extraction rate. The highest protein extraction rate was achieved at a hydrolysis temperature of 37℃. Therefore, the preferred hydrolysis temperature for this experimental example is 37℃.

[0050] 3. Screening results for enzymatic hydrolysis pH The results are as follows Figure 2 As shown in Figure C. The results indicate that the pH of the enzymatic hydrolysis reaction has a significant impact on the protein extraction rate. The highest protein extraction rate was achieved at a pH of 6. Therefore, the preferred enzymatic hydrolysis pH in this experimental example is 6.

[0051] 4. Screening results of compound enzyme addition amount The results are as follows Figure 2 As shown in Figure D. The results indicate that the amount of compound enzyme added has a significant impact on the protein extraction rate. In particular, when the amount of compound enzyme added is 3%, the protein extraction rate decreases significantly. Among them, the protein extraction rate is highest when the amount of compound enzyme added is 4-5%. Therefore, the preferred amount of compound enzyme added in this experimental example is 4-5%.

[0052] 5. Screening results of solid-liquid ratio The results are as follows Figure 2 As shown in E. The results showed that the solid-liquid ratio of mycelial powder to water had a significant impact on the protein extraction rate. Among them, the protein extraction rate was the highest when the solid-liquid ratio was 1:50. Therefore, the preferred solid-liquid ratio of mycelial powder to water in this experimental example was 1:50.

[0053] 6. Screening results of enzymatic hydrolysis time The results are as follows Figure 2 As shown in F. The results indicate that the enzymatic hydrolysis time has a significant impact on the protein extraction rate. The highest protein extraction rate was achieved when the enzymatic hydrolysis time was 2-3 hours. Therefore, the preferred enzymatic hydrolysis time in this experimental example is 2-3 hours.

[0054] The above results indicate that the optimal enzymatic hydrolysis process was obtained in this experiment: the ratio of the compound enzyme (Driselase:Lywallzyme:Zymolyase:Snailase) was 1:1:2:1, the hydrolysis temperature was 37℃, the hydrolysis pH was 6, the amount of compound enzyme added was 4-5%, the solid-liquid ratio of mycelial dry powder to water was 1:50, and the hydrolysis time was 2-3h.

[0055] Experiment Example 2: Screening Experiment of Alkali Extraction Scheme I. Experimental Methods 1. Screening experiment on alkali extraction time Protein powder was prepared according to the method in Example 1, wherein the alkaline extraction times in step S3 were 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h, respectively. The protein extraction rate of each group was measured.

[0056] 2. Screening experiment for alkali extraction temperature Protein powder was prepared according to the method in Example 1, wherein in step S3, the alkaline extraction temperatures were 30℃, 40℃, 50℃, and 60℃, respectively. The protein extraction rate of each group was measured.

[0057] 3. Screening experiment for alkali concentration Protein powder was prepared according to the method in Example 1, wherein the final concentrations of NaOH in step S3 were 20 mM, 30 mM, 40 mM, 50 mM, and 60 mM, respectively. The protein extraction rate of each group was measured.

[0058] 4. Determination of protein extraction rate Protein extraction rate = mass of protein obtained from alkaline extraction and acid precipitation / total mass of protein in raw material × 100%.

[0059] II. Experimental Results 1. Screening results of alkali extraction time The results are as follows Figure 3 As shown in Figure A, the results indicate that the alkaline extraction time has a significant impact on the protein extraction rate. The highest protein extraction rate was observed when the alkaline extraction time was 1.5 hours. Therefore, the preferred alkaline extraction time in this experimental example is 1.5 hours.

[0060] 2. Screening results of alkali extraction temperature The results are as follows Figure 3 As shown in B. The results show that the alkaline extraction temperature has a significant impact on the protein extraction rate. Among them, the protein extraction rate is higher when the alkaline extraction temperature is 40-60℃. Therefore, the preferred alkaline extraction temperature in this experimental example is 40-60℃, and more preferably 40℃.

[0061] 3. Screening results for alkali concentration The results are as follows Figure 3 As shown in Figure C. The results indicate that the concentration of alkali has a significant impact on the protein extraction rate. The highest protein extraction rate was achieved when the alkali concentration was 50 mM. Therefore, the preferred alkali concentration in this experimental example is 50 mM.

[0062] The above results indicate that the optimal alkali extraction process parameters were obtained in this experimental example: alkali extraction time of 1.5 h, alkali extraction temperature of 40-60℃, and alkali concentration of 50 mM.

[0063] Experiment Example 3: Screening Experiment of Ultrasonic-Assisted Extraction Scheme I. Experimental Methods 1. Screening experiment for ultrasonic power density Protein powder was prepared according to the method in Example 1, wherein in step S4, the ultrasonic power density was 200 W / L, 300 W / L, 400 W / L, and 500 W / L, respectively. The protein extraction rate of each group was measured.

[0064] 2. Screening experiment for ultrasound time Protein powder was prepared according to the method in Example 1, wherein in step S4, the ultrasonic treatment time was 5 min, 10 min, 15 min, 20 min, and 30 min, respectively. The protein extraction rate of each group was measured.

[0065] 3. Determination of protein extraction rate Same as Experiment 2.

[0066] II. Experimental Results 1. Screening results of ultrasonic power density The results are as follows Figure 4 As shown in Figure A. The results indicate that ultrasonic power density has a significant impact on protein extraction rate. The highest protein extraction rate was achieved when the ultrasonic power density was 300-400 W / L. Therefore, the preferred ultrasonic power density in this experimental example is 300-400 W / L.

[0067] 2. Screening results for ultrasound time The results are as follows Figure 4 As shown in B. The results showed that the protein extraction rate was higher when the sonication time was 10 min or 30 min. Therefore, the preferred sonication time in this experimental example was 10 min or 30 min.

[0068] The above results indicate that the optimal ultrasound-assisted extraction process was obtained in this experiment: the ultrasound power density was 300-400 W / L, and the ultrasound time was 10 min or 30 min.

[0069] Experiment Example 4: Yield and emulsifying properties of Fusarium venetum TB01 protein. I. Experimental Methods 1. Protein yield and purity The Kjeldahl method (GB 5009.5-2025) was adopted for nitrogen determination.

[0070] 2. Determination of crude protein content Crude protein content (%) = mass of protein in product obtained by alkali extraction and acid precipitation / total mass of raw materials × 100%.

[0071] 3. Emulsifying properties All protein samples were compared with commercially available whey protein isolate (WP, catalog number: S55564) and soy protein isolate (SP, catalog number: S30914) from Yuanye Biotechnology Co., Ltd. All test samples were prepared as 0.5 wt% aqueous solutions (pH 7.0), and the emulsifying activity index (EAI) and emulsifying stability index (ESI) were determined. The methods for determining EAI and ESI are as follows: Take 50 µL of each of the freshly prepared emulsion and the emulsion sample stored for 10 minutes, respectively, from the bottom of the test tube, and add them to 5 mL of 0.1% SDS solution. After vortex mixing, measure the absorbance at 500 nm. The emulsion activity index (EAI) and emulsion stability index (ESI) are calculated according to the following formulas: EAI (m 2 / g) = (2×2.303×A0×N) / (C×φ×10000) ESI (min) = A0 / (A0 - A 10 ) × t Among them, A0 and A 10The values ​​represent the absorbance at 500 nm at 0 minutes and 10 minutes, respectively; N is the dilution factor (100); C is the protein concentration in the aqueous phase before emulsification (g / mL); φ is the volume fraction of the oil phase (0.05); and t is the time interval (10 minutes).

[0072] II. Experimental Results 1. Protein sample properties Visual images of the test protein emulsion, such as Figure 5 As shown in Table 1, the performance test results of the protein samples are as follows. The protein yield, emulsifying activity, and stability of the product obtained in Example 1 (using 50 mM NaOH, 40 ℃, 1.5 h) were all higher than those in Example 2 (30 mM, 40 ℃, 1.5 h) and Example 3 (60 mM, 40 ℃, 1.5 h). This result is consistent with... Figure 2 The optimization trends shown are consistent, indicating that changes in alkaline extraction conditions have a clear impact on protein extraction efficiency and functional properties.

[0073] Table 1 Product Performance Comparison Table Comparing the experimental data of Example 1 and Comparative Example 1 (without ultrasonic treatment), it can be seen that under the same pretreatment conditions, omitting the ultrasonic step significantly reduces the protein yield, emulsification activity index, and emulsification stability, proving that ultrasonic treatment plays a necessary role in improving the protein extraction rate and its functional properties.

[0074] Compared with Comparative Example 2 (conventional alkali extraction and acid precipitation method), Example 1 showed significant advantages in all indicators, demonstrating the overall progress of the process of the present invention.

[0075] The emulsifying activity index of product FV1 is 242.46 m 2 The emulsification activity index ( / g) and emulsification stability (54.92 min) of product FV1 are both higher than those of commercially available soy protein SP; moreover, the emulsification activity index of product FV1 is similar to that of commercially available whey protein WP, and its emulsification stability is significantly higher than that of WP.

[0076] The above results indicate that by pretreatment with enzymatic hydrolysis to break the cell wall, optimizing alkaline extraction conditions, and introducing ultrasonic treatment, the process of this invention can effectively extract and modify Fusarium venetum TB01 protein, making its emulsification stability higher than that of whey protein and soy protein.

[0077] Example 5: Physicochemical properties of Fusarium vesicatoria TB01 protein This experiment determined the physicochemical properties of FV1 prepared in Example 1, commercially available whey protein isolate (WP), and commercially available soy protein isolate (SP).

[0078] I. Experimental Methods 1. Determination of average particle size, PDI and surface charge The average particle size, PDI (polydispersity index), and surface charge were determined using a dynamic light scattering nanoparticle size potentiometer. The sample concentration was 0.1% wt, and the pH of the sample solution was 7.0.

[0079] 2. Determination of protein solubility Protein solubility was determined using the BCA method with a sample concentration of 0.1% wt and a sample solution pH of 7.0.

[0080] II. Experimental Results The experimental results are shown in Table 2.

[0081] Table 2. Comparison of physicochemical properties between the protein of this invention and commercially available proteins. Note: The superscripts a, b, and c indicate that the significance analysis result of the same test indicator is p<0.05.

[0082] Physicochemical analysis of the protein FV1 of this invention showed that its average particle size was 1387.00 nm, significantly smaller than that of commercially available soy protein SP and close to that of commercially available whey protein WP, indicating that it has a small aggregate size in the aqueous phase. The Zeta potential of FV1 was -38.47 mV, which was higher than that of WP (-29.47 mV) and SP (-31.80 mV), indicating that it carried a stronger net negative charge on its surface at pH 7.0, which helps maintain the colloidal stability of the system through electrostatic repulsion. Furthermore, the solubility of FV1 under neutral conditions was 76.46%, significantly higher than that of WP (65.56%) and SP (32.61%), indicating that it has better dispersibility and availability in the aqueous phase.

[0083] The above results indicate that, compared to commercially available whey protein isolate (WP) and commercially available soy protein isolate (SP), FV1 exhibits a stronger negative surface charge, higher solubility, and smaller hydrated particle size in aqueous solution. This combination of fundamental physicochemical properties suggests that FV1 holds promise for demonstrating good functionality in applications such as emulsification.

[0084] Experimental Example 6: Determination of the properties of Fusarium vesicatoria TB01 protein at the oil-water interface I. Experimental Methods This experiment measured the emulsifying abilities of FV1 prepared in Example 1, commercially available whey protein isolate (WP), and commercially available soy protein isolate (SP). The dynamic changes in surface tension (γ, mN / m) at the oil-water interface for the three samples (WP, SP, and FV1) over time (0–7200 s) were also measured. The methods were as follows: The interfacial adsorption behavior of the protein at the oil-water interface was characterized using an interfacial rheometer (Tracker, TECLIS, France). Purified medium-chain triglycerides (MCT) were used as the oil phase, and the protein aqueous solution was diluted with deionized water to a final concentration of 1 mg / mL. Interfacial tension was determined by fitting the droplet profile captured by a charge-coupled device (CCD) camera to a numerical model. The adsorption process was continuously monitored for 7200 s to obtain the equilibrium value. All measurements were performed at 25 °C.

[0085] II. Experimental Results Experimental results of oil-water interface properties are as follows: Figure 6 As shown in the figure, the test results indicate that the three samples exhibit similar adsorption kinetics. In the initial stage (0–2000 s), the interfacial tension of all three decreased rapidly from its initial value, indicating that protein molecules rapidly migrated to and adsorbed at the oil-water interface; subsequently, the rate of decrease slowed down, eventually reaching dynamic equilibrium. SP exhibited the highest interfacial tension value throughout the process, with both its initial and equilibrium values ​​significantly higher than those of FV1 and WP. The kinetic curves of FV1 and WP highly overlapped, with their initial and equilibrium interfacial tensions at the same level.

[0086] Test results show that, compared with SP, FV1 and WP are comparable in terms of the kinetic process of reducing oil-water interfacial tension, adsorption efficiency, and the final achievable interfacial tension level. They can more effectively replace water molecules on the interface, reduce the interfacial energy of the system, and have higher emulsification ability and emulsification stability.

[0087] The results of this experiment provide direct evidence from the perspective of interface properties that the FV1 protein of this invention possesses high emulsifying ability.

[0088] As can be seen from the above embodiments and experimental examples, this invention provides a highly emulsifying Fusarium venetum TB01 protein, its preparation method, and its applications. This invention uses Fusarium venetum TB01 mycelium as raw material and innovatively combines enzymatic hydrolysis and ultrasonic treatment in a sequential and synergistic manner. This achieves efficient cell wall disruption and extraction of a high-content protein (≥82%) while simultaneously functionalizing the protein structure. Testing showed that the obtained protein exhibited significantly better emulsifying activity and stability than soy protein, and its emulsifying stability was superior to commercial whey protein. Therefore, the protein obtained by this invention can be used as a high-performance emulsifying ingredient to replace or partially replace whey protein or soy protein in food systems such as beverages, plant-based yogurt, and sauces.

Claims

1. A type of Venetian Fusarium ( Fusarium venenatum TB01 protein, characterized in that, The Venetian Fusarium TB01 protein was prepared by the following steps: Step 1: Fusarium vesicatoria TB01 is mixed with cell wall lysin for enzymatic hydrolysis. Step 2: The enzymatic hydrolysis product is extracted with alkali to obtain an alkali extract, wherein the concentration of alkali is 40-60 mM and the alkali extraction temperature is 40-60℃. Step 3: Sonicate the alkaline extract at a power density of 300-400 W / L, then add acid to obtain the final product. In step 1, the cell wall lysing enzyme is a complex enzyme composed of lysis enzyme, fungal cell wall lysing enzyme, yeast cell wall lysing enzyme, and snail enzyme, with a mass ratio of 1-2:1-2:1-2:1-2; The conditions for the enzymatic hydrolysis reaction include: a temperature of 37-40℃, a pH of 6-6.5, and a reaction time of 2-3 hours. Before mixing with cell wall dissolving enzymes, Fusarium moniliforme TB01 is pre-dissolved in water. The mass ratio of Fusarium moniliforme TB01 dry powder to water is 1:40-60. The amount of cell wall dissolving enzyme added is 2% or 4% to 6% of the dry basis; In step 2, the alkali is an inorganic strong alkali, and the alkali extraction conditions include: extraction time of 0.5-2 hours; In step 3, the ultrasound time is 5-30 minutes; The specific process of adding acid includes: adjusting the pH to 3.6-4.0, and letting it stand at 4-6 ℃ for 8-12 hours; The acid is an inorganic strong acid; after the acid addition operation, centrifugation, washing, alkali neutralization, and drying are performed.

2. The method for preparing Fusarium vesicatoria TB01 protein according to claim 1, characterized in that, It includes the following steps: Step 1: Fusarium vesicatoria TB01 is mixed with cell wall lysin for enzymatic hydrolysis. Step 2: The enzymatic hydrolysis product is extracted with alkali to obtain an alkali extract, wherein the concentration of alkali is 40-60 mM and the alkali extraction temperature is 40-60℃. Step 3: Sonicate the alkaline extract at a power density of 300-400 W / L, then add acid to obtain the final product. In step 1, the cell wall lysing enzyme is a complex enzyme composed of lysis enzyme, fungal cell wall lysing enzyme, yeast cell wall lysing enzyme, and snail enzyme, with a mass ratio of 1-2:1-2:1-2:1-2; The conditions for the enzymatic hydrolysis reaction include: a temperature of 37-40℃, a pH of 6-6.5, and a reaction time of 2-3 hours. Before mixing with cell wall dissolving enzymes, Fusarium moniliforme TB01 is pre-dissolved in water. The mass ratio of Fusarium moniliforme TB01 dry powder to water is 1:40-60. The amount of cell wall dissolving enzyme added is 2% or 4% to 6% of the dry basis; In step 2, the alkali is an inorganic strong alkali, and the alkali extraction conditions include: extraction time of 0.5-2 hours; In step 3, the ultrasound time is 5-30 minutes; The specific process of adding acid includes: adjusting the pH to 3.6-4.0, and letting it stand at 4-6 ℃ for 8-12 hours; The acid is an inorganic strong acid; after the acid addition operation, centrifugation, washing, alkali neutralization, and drying are performed.

3. The use of the Fusarium TB01 protein of claim 1 in the preparation of protein foods as an emulsifying ingredient.

4. A low-fat protein food, characterized in that: It includes the Fusarium vesicatoria TB01 protein as described in claim 1.

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