Highly digestible yeast protein and process for its preparation and use

Abstract

The application discloses high-digestibility yeast protein and a preparation method and application thereof. First, ordinary yeast protein powder is prepared into a yeast protein dispersion, then the protein structure and aggregation state are regulated by two-stage ultrasonic treatment under temperature control, and then glutaminase and beta-glucanase are sequentially added for enzymatic modification, thereby obtaining high-digestibility yeast protein. By the modification strategy of two-stage ultrasonic pretreatment and sequential double-enzyme treatment, the comprehensive regulation of the aggregation state, molecular conformation and polysaccharide barrier effect of the yeast protein is realized, and the overall method has good technical effect and application value. The method combines physical modification and enzymatic modification, disperses, depolymerizes and restructures the protein by two-stage ultrasonic treatment, and then adjusts the protein conformation and polysaccharide barrier by glutaminase and beta-glucanase respectively.

Description

Technical Field

[0001] This invention relates to the field of food protein processing, specifically to a yeast protein with high digestibility, its preparation method, and its uses. Background Technology

[0002] Yeast protein, as a microbial protein, possesses advantages such as stable source, short production cycle, low resource consumption, and relatively balanced amino acid composition, making it promising for applications in food processing, nutritional supplementation, and the development of emergency alternative proteins. Compared to traditional animal proteins and some plant proteins, yeast protein offers advantages in large-scale production, sustainable raw material availability, and adaptability to storage and transportation, thus attracting widespread attention. However, existing yeast proteins still face certain limitations in practical applications. On one hand, yeast protein molecules tend to form strong aggregates, and some structures are quite dense, resulting in poor dispersibility and solubility in aqueous phases, insufficient exposure of digestive enzyme action sites, and thus limiting their digestibility and absorption efficiency. On the other hand, yeast-derived raw materials often contain cell walls and related polysaccharide components, which can hinder protein release and the contact between digestive enzymes and substrates, further affecting the nutritional utilization of yeast protein.

[0003] To improve the functional properties and digestibility of yeast proteins, current research primarily employs ultrasound, enzymatic hydrolysis, or other single modification methods. Ultrasound treatment promotes particle dispersion and structural loosening through cavitation and shearing, while enzymatic treatment can further regulate protein conformation or weaken related polysaccharide barriers. However, single-treatment methods often have limitations. For example, single-stage ultrasound treatment may suffer from insufficient energy utilization or excessively strong local effects, making it difficult to simultaneously achieve dispersion, depolymerization, and structural reconstruction; single enzymatic treatment typically only targets a specific type of limiting factor in the system, making it difficult to simultaneously achieve protein structure regulation and polysaccharide barrier weakening. Therefore, existing methods still have room for further optimization in improving yeast protein solubility and in vitro simulated digestion performance. Summary of the Invention

[0004] The purpose of this invention is to provide a yeast protein with high digestibility, its preparation method, and its uses. This invention first prepares a yeast protein dispersion from ordinary yeast protein powder. Then, under controlled temperature conditions, a two-stage ultrasonic treatment is used to regulate the protein structure and aggregation state. Subsequently, glutaminase and β-glucanase are added sequentially to perform enzymatic modification, resulting in a yeast protein with high digestibility.

[0005] The objective of this invention is achieved through the following technical solution: A method for preparing highly digestible yeast protein includes the following steps: (1) Disperse yeast protein powder in water and perform the first stage of ultrasonic treatment. The ultrasonic frequency is 15~20 kHz, the ultrasonic power is 100~350 W, and the treatment time is 3~10 min. Then perform the second stage of ultrasonic treatment. The ultrasonic frequency is 15~20 kHz, the ultrasonic power is 400~600 W, and the treatment time is 3~10 min to obtain the ultrasonic treatment solution. Both ultrasonic treatments are performed in pulse mode. The pulse parameters are 3~5 s on and 3~5 s off. (2) Adjust the pH of the ultrasonic treatment solution obtained in step (1) to 7.0~8.0, add glutaminase, and hydrolyze at 45~55 ℃ for 4~6 h; then adjust the pH of the hydrolysis system to 4.0~5.0, add β-glucanase, and hydrolyze at 45~55 ℃ for 5~8 h. After the hydrolysis is completed, inactivate the enzyme to obtain a hydrolysate containing yeast protein with high digestibility. The yeast protein powder mentioned in step (1) is commercially available food-grade yeast protein powder, which is made from edible yeast cells through cell wall breaking, protein extraction, separation and drying.

[0006] Preferably, the ultrasonic power in the first stage of step (1) is 150~250 W, more preferably 180 W, and the processing time is more preferably 5 min; the ultrasonic power in the second stage is 450~550 W, more preferably 520 W, and the processing time is more preferably 5 min. In step (1), the system temperature must be kept below 30 °C during both ultrasonic treatment processes; In step (2), the glutaminase is a food-grade enzyme, and its addition amount is 0.05~0.5% of the yeast protein powder mass, preferably 0.1~0.3%; In step (2), β-glucanase is a food-grade enzyme, and its addition amount is 0.2-1.0% of the mass of yeast protein powder, preferably 0.5-0.8%; In step (2), the enzyme inactivation conditions are to keep the temperature at 90-95 °C for 15-30 min. The enzymatic hydrolysate containing highly digestible yeast protein described in step (2) can be further concentrated and spray-dried to obtain highly digestible yeast protein powder; The concentration is preferably carried out to a dry matter content of 20-25%. The vacuum concentration temperature shall not exceed 55 ℃; The outlet air temperature of the spray dryer shall not exceed 90 ℃.

[0007] The highly digestible yeast protein obtained by the above method can be used to prepare functional foods, fortified foods, and foods for special medical purposes.

[0008] The beneficial effects of this invention are reflected in the following aspects: 1. This invention employs a two-stage ultrasonic treatment to regulate the yeast protein system stepwise, achieving clear staged action targets and better modification effects. The first stage uses relatively low-intensity ultrasonic treatment, primarily to promote yeast protein dispersion, reduce large particle aggregates, and achieve initial deagglomeration, thereby improving system homogeneity and establishing a good reaction foundation for subsequent treatments. The second stage uses relatively high-intensity ultrasonic treatment, further loosening and reconstructing the protein molecular structure based on the thorough dispersion in the first stage. This opens up some compact conformations, exposing more potential enzyme action sites and hydrophilic groups. Compared to single-stage continuous ultrasonic treatment, the two-stage ultrasonic treatment method of this invention, while ensuring the modification effect, reduces local heat accumulation, excessive cavitation, structural damage, and secondary aggregation that may be caused by continuous high-intensity treatment. It is more conducive to maintaining the stability of the protein system, improving the reproducibility of the treatment results, and providing a more suitable substrate state for subsequent enzymatic digestion.

[0009] 2. This invention employs a sequential treatment approach using glutaminase and β-glucanase to regulate yeast proteins in stages, exhibiting significant synergistic modification advantages. First, glutaminase acts on the yeast protein system after two stages of ultrasonic pretreatment to regulate the charge distribution and spatial conformation of protein molecules, improving protein dispersion and solubility, ultimately enhancing the accessibility of subsequent enzyme action and digestibility. Subsequently, β-glucanase degrades the residual polysaccharide components of the yeast protein, mitigating the adverse effects of the polysaccharide barrier on protein release and subsequent digestion. This sequential treatment approach targets two different objectives—protein conformation regulation and polysaccharide barrier weakening—each working in turn, reducing the impact of cross-targeting of enzymes, thus improving overall modification efficiency and enhancing the digestibility of yeast proteins.

[0010] 3. This invention employs a two-stage ultrasonic pretreatment combined with sequential dual-enzyme treatment modification strategy to achieve comprehensive regulation of yeast protein aggregation state, molecular conformation, and polysaccharide barrier effect. The overall method exhibits good technical efficacy and application value. This method combines physical modification with enzymatic modification. First, two-stage ultrasonic treatment achieves protein dispersion, depolymerization, and structural reconstruction. Then, glutaminase and β-glucanase are used to specifically regulate protein conformation and the polysaccharide barrier, respectively. Compared with ultrasonic treatment alone, single-enzyme treatment, or single-stage ultrasonic combined with enzyme treatment, the method of this invention is more conducive to improving the digestibility of yeast proteins. Furthermore, the process conditions used in this invention are relatively mild, do not introduce toxic or harmful chemical reagents, and possess good safety and green processing characteristics. It also has good process controllability and scale-up application potential, and can be widely applied to the development of protein-fortified foods, functional food ingredients, beverage products, powder products, and other protein-based ingredients. Attached Figure Description

[0011] Figure 1 Fourier transform infrared spectra of multi-stage ultrasonically modified yeast protein and untreated yeast protein; Figure 2 The ζ-potential analysis diagrams are shown for multi-stage ultrasonically modified yeast protein, single-stage ultrasonically modified yeast protein, and untreated yeast protein. Figure 3 The average particle size analysis diagrams are shown for multi-stage ultrasonically modified yeast protein, single-stage ultrasonically modified yeast protein, and untreated yeast protein. The untreated yeast protein mentioned above is commercially available food-grade yeast protein powder, which is made from edible yeast cells through cell wall breaking, protein extraction, separation and drying. The multi-stage ultrasonically modified yeast protein is the ultrasonic treatment solution A obtained in step (2) of Example 1; The single-stage ultrasonically modified yeast protein is the ultrasonic treatment solution 2 obtained in step (2) of Comparative Example 2. Detailed Implementation

[0012] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0013] The methods for determining the test indicators involved in the embodiments of the present invention are as follows: 1. Solubility determination method In this invention, the solubility of the yeast protein dispersion system after ultrasonic treatment was determined using the Kjeldahl method. Specifically, after thoroughly mixing the sample to be tested, 2.0 ± 0.01 g of the sample was weighed and added to a digestion tube for determining the total nitrogen content (denoted as N). total Separate samples from the same batch were centrifuged at 4 ℃, 8000 × g, for 10 min, and the supernatant was collected. Subsequently, 2.0 ± 0.01 g of the supernatant was weighed and added to a digestion tube for the determination of soluble nitrogen content (denoted as N). sol Nitrogen content determination was performed according to the Kjeldahl nitrogen determination procedure: 10 mL of concentrated sulfuric acid and a Kjeldahl catalyst were added to the digestion tube and heated until clear. After cooling, sodium hydroxide solution was added for alkalization, followed by distillation to allow ammonia to be distilled off and absorbed into boric acid solution. The nitrogen content was then calculated using 0.1 mol / L hydrochloric acid solution and blank correction. Solubility (characterized by nitrogen solubility index, %) was calculated using the following formula: Where N sol The nitrogen content of the supernatant sample, N total This represents the total nitrogen content of the original sample. Each sample group should be measured in parallel at least three times.

[0014] 2. In vitro simulated digestibility assay method In this invention, the in vitro simulated digestion of modified yeast protein was evaluated using the INFOGEST 2.0 static in vitro digestion model. The simulated digestion solution was prepared according to the INFOGEST 2.0 standard formula (including electrolyte composition and CaCl2 addition, etc.). In this invention, the oral cavity stage was omitted, and only the gastric and intestinal stages were simulated for digestion. The nitrogen content before and after digestion was determined by the Kjeldahl method to calculate its simulated in vitro digestibility. First, after thoroughly mixing the sample to be tested, weigh 2.0 ± 0.01 g of the sample and add it to a digestion tube to determine the total nitrogen content before digestion (denoted as N0). Separately, take samples from the same batch and perform simulated digestion in the stomach and intestine stages according to the INFOKE 2.0 method. In the stomach stage, the sample and simulated gastric juice are mixed at a volume ratio of 1:1. The pH of the mixture is adjusted to 3.0 ± 0.1 using 0.5 mol / L hydrochloric acid, and the mixture is incubated at 37 ℃ with shaking for 120 min. The pepsin activity in the simulated gastric juice is 4000 U / mL, and after mixing it with the sample at a 1:1 ratio, the final pepsin activity of the system is 2000 U / mL. The intestinal stage begins with gastric digestive fluid, which is mixed with simulated intestinal fluid at a 1:1 volume ratio. The pH of the mixture is adjusted to 7.0 ± 0.1 using 0.5 mol / L sodium hydroxide, and incubated with shaking at 37 ℃ for 120 min. The trypsin activity in the simulated intestinal fluid is 200 U / mL. After mixing with the gastric digestive fluid at a 1:1 ratio, the final trypsin activity is 100 U / mL. Immediately after digestion, the pH is adjusted to 3.0 ± 0.1 using 0.5 mol / L HCl to inactivate intestinal trypsin. The sample is then centrifuged at 4 ℃, a centrifugation rate of 10000 × g, and a centrifugation time of 10 min. The supernatant is collected, and 2.0 ± 0.01 g is weighed for determining the soluble nitrogen content (denoted as N) after digestion. d The nitrogen content determination was performed according to the Kjeldahl nitrogen determination procedure: 10 mL of concentrated sulfuric acid and Kjeldahl catalyst were added to the digestion tube and heated until clear. After cooling, alkali was added and distilled to distill off ammonia, which was then absorbed into a boric acid solution. Subsequently, the nitrogen content was calculated by titration with 0.1 mol / L hydrochloric acid solution and blank correction. The simulated in vitro digestibility (%) was calculated using the following formula: Where N0 is the total nitrogen content of the sample before digestion, N d To determine the nitrogen content of the supernatant sample at the end of digestion, f This refers to the volume dilution factor during the digestion process.

[0015] Example 1 A method for preparing a yeast protein powder with high digestibility includes the following steps: (1) Weigh 60.0±0.1 g of yeast protein powder, add it to 940.0 g of deionized water, and disperse it evenly under rapid stirring to prepare a yeast protein dispersion with a solid content of about 6%.

[0016] The yeast protein powder used as a raw material in this invention is a commercially available food-grade yeast protein powder, which is obtained from edible yeast cells through cell wall disruption, protein extraction, separation, and drying. The same applies below.

[0017] (2) The yeast protein dispersion was placed in an ice bath and an ultrasonic probe was inserted for two-stage ultrasonic treatment. The ultrasonic frequency was 20 kHz, and a pulse mode was used with pulse parameters of 3 s on and 3 s off. The ultrasonic power of the first stage was 180 W, and the treatment time was 5 min; the ultrasonic power of the second stage was 520 W, and the treatment time was 5 min. During the ultrasonic treatment, the system temperature was controlled to not exceed 30 ℃ by using an ice bath. After the treatment, ultrasonic treatment solution A was obtained.

[0018] (3) Adjust the pH of the ultrasonic treatment solution A to 8.0, add 0.3 g of glutaminase (0.5% of yeast protein content), and hydrolyze at 55 °C for 4 h; then adjust the pH of the hydrolysis system to 5.0, add 0.6 g of β-glucanase (1.0% of yeast protein content), and hydrolyze at 55 °C for 5 h. After hydrolysis, inactivate the enzyme by incubating at 95 °C for 15 min to obtain modified yeast protein hydrolysate A.

[0019] (4) Vacuum concentrate the modified yeast protein hydrolysate A to a concentration of 25%, and then spray dry it to obtain modified yeast protein powder A.

[0020] The in vitro simulated gastrointestinal digestibility results of modified yeast protein powder A are shown in Table 1, and the solubility results of ultrasonically treated solution A are shown in Table 2.

[0021] Example 2 A method for preparing a yeast protein powder with high digestibility includes the following steps: (1) Weigh 60.0±0.1 g of yeast protein powder, add it to 940.0 g of deionized water, and disperse it evenly under rapid stirring to prepare a yeast protein dispersion with a solid content of about 6%.

[0022] (2) The yeast protein dispersion was placed in an ice bath and an ultrasonic probe was inserted for two-stage ultrasonic treatment. The ultrasonic frequency was 20 kHz, and a pulse mode was used with pulse parameters of 3 s on and 3 s off. The ultrasonic power of the first stage was 150 W, and the treatment time was 3 min; the ultrasonic power of the second stage was 450 W, and the treatment time was 3 min. During the ultrasonic treatment, the system temperature was controlled to not exceed 30 ℃ by using an ice bath. After the treatment, ultrasonically treated solution B was obtained.

[0023] (3) Adjust the pH of the ultrasonic treatment solution B to 8.0, add 0.12 g of glutaminase (0.2% of yeast protein content), and hydrolyze at 55 °C for 4 h; then adjust the pH of the hydrolysis system to 5.0, add 0.36 g of β-glucanase (0.6% of yeast protein content), and hydrolyze at 55 °C for 5 h. After hydrolysis, inactivate the enzyme by incubating at 95 °C for 15 min to obtain modified yeast protein hydrolysate B.

[0024] (4) Vacuum concentrate the modified yeast protein hydrolysate B to a concentration of 25%, and then spray dry it to obtain modified yeast protein powder B.

[0025] The in vitro simulated gastrointestinal digestibility results of modified yeast protein powder B are shown in Table 1, and the solubility results of ultrasonically treated solution B are shown in Table 2.

[0026] Example 3 A method for preparing a yeast protein powder with high digestibility includes the following steps: (1) Weigh 60.0±0.1 g of yeast protein powder, add it to 940.0 g of deionized water, and disperse it evenly under rapid stirring to prepare a yeast protein dispersion with a solid content of about 6%.

[0027] (2) The yeast protein dispersion was placed in an ice bath and an ultrasonic probe was inserted for two-stage ultrasonic treatment. The ultrasonic frequency was 20 kHz, and a pulse mode was used with pulse parameters of 3 s on and 3 s off. The ultrasonic power of the first stage was 250 W, and the treatment time was 10 min; the ultrasonic power of the second stage was 550 W, and the treatment time was 10 min. During the ultrasonic treatment, the system temperature was controlled to not exceed 30 ℃ by using an ice bath. After the treatment, ultrasonic treatment solution C was obtained.

[0028] (3) Adjust the pH of the ultrasonic treatment solution C to 8.0, add 0.03 g of glutaminase (0.05% of yeast protein content), and hydrolyze at 55 °C for 4 h; adjust the pH of the hydrolysis system to 5.0, add 0.12 g of β-glucanase (0.2% of yeast protein content), and hydrolyze at 55 °C for 5 h. After hydrolysis, inactivate the enzyme by incubating at 95 °C for 15 min to obtain modified yeast protein hydrolysate C.

[0029] (4) Vacuum concentrate the modified yeast protein hydrolysate C to a concentration of 25%, and then spray dry it to obtain modified yeast protein powder C.

[0030] The in vitro simulated gastrointestinal digestibility of modified yeast protein powder C is shown in Table 1, and the solubility of ultrasonically treated solution C is shown in Table 2.

[0031] Comparative Example 1 A method for preparing yeast protein powder, which differs from Example 1 in that: ultrasonic treatment is not performed, while the other conditions are the same as in Example 1, including the following steps: (1) Same as step (1) in Example 1.

[0032] (2) The yeast protein dispersion was placed in an ice bath and left to stand for 10 min without ultrasonic treatment to obtain untreated yeast protein dispersion 1.

[0033] (3) The untreated yeast protein dispersion 1 was enzymatically hydrolyzed, and the operation was the same as step (3) in Example 1, to obtain modified yeast protein hydrolysate 1.

[0034] (4) Same as step (4) in Example 1, to obtain modified yeast protein powder 1.

[0035] The in vitro simulated gastrointestinal digestibility of modified yeast protein powder 1 is shown in Table 1, and the solubility of untreated yeast protein dispersion is shown in Table 2.

[0036] Comparative Example 2 A method for preparing yeast protein powder, which differs from Example 1 in that the two-stage ultrasonic treatment is replaced with a single-stage ultrasonic treatment, while the other conditions are the same as in Example 1, including the following steps: (1) Same as step (1) in Example 1.

[0037] (2) The yeast protein dispersion was placed in an ice bath and an ultrasonic probe was inserted for single-stage ultrasonic treatment. The ultrasonic frequency was 20 kHz, and the pulse mode was used with pulse parameters of 3 s on and 3 s off. The ultrasonic power was 520 W and the treatment time was 5 min. During the ultrasonic treatment, the system temperature was controlled to not exceed 30 ℃ by using an ice bath. After the treatment, ultrasonic treatment solution 2 was obtained.

[0038] (3) Enzymatically hydrolyze the ultrasonic treatment solution 2, following the same procedure as step (3) in Example 1, to obtain modified yeast protein hydrolysate 2.

[0039] (4) Same as step (4) in Example 1, to obtain modified yeast protein powder 2.

[0040] The in vitro simulated gastrointestinal digestibility results of modified yeast protein powder 2 are shown in Table 1, and the solubility results of ultrasonically treated liquid 2 are shown in Table 2.

[0041] Comparative Example 3 A method for preparing yeast protein powder, which differs from Example 1 in that: only β-glucanase is used for enzymatic hydrolysis, without the addition of glutaminase, while the other conditions are the same as in Example 1, including the following steps: (1) Same as step (1) in Example 1.

[0042] (2) Same as step (2) in Example 1.

[0043] (3) Adjust the pH of the two-stage ultrasonic treatment solution to 5.0, add 0.48 g of β-glucanase (0.8% of yeast protein content), and enzymatically hydrolyze at 55 °C for 5 h. After enzymatic hydrolysis, inactivate the enzyme by incubating at 95 °C for 15 min to obtain modified yeast protease hydrolysate 3.

[0044] (4) Same as step (4) in Example 1, to obtain modified yeast protein powder 3.

[0045] The in vitro simulated gastrointestinal digestibility results of modified yeast protein powder 3 are shown in Table 1.

[0046] Comparative Example 4 A method for preparing yeast protein powder, which differs from Example 1 in that: only glutaminase is used for enzymatic hydrolysis, without the addition of β-glucanase, while the other conditions are the same as in Example 1, including the following steps: (1) Same as step (1) in Example 1.

[0047] (2) Same as step (2) in Example 1.

[0048] (3) Adjust the pH of the two-stage ultrasonic treatment solution to 8.0, add 0.18 g of glutaminase (0.3% of yeast protein content), and hydrolyze at 55 °C for 4 h. After hydrolysis, inactivate the enzyme by incubating at 95 °C for 15 min to obtain modified yeast protein hydrolysate 4.

[0049] (4) Same as step (4) in Example 1, to obtain modified yeast protein powder 4.

[0050] The in vitro simulated gastrointestinal digestibility results of modified yeast protein powder 4 are shown in Table 1.

[0051] Comparative Example 5 A method for preparing yeast protein powder, which differs from Example 1 in that the order of the two-stage ultrasound is adjusted, while the other conditions are the same as in Example 1, including the following steps: (1) Same as step (1) in Example 1.

[0052] (2) The yeast protein dispersion was placed in an ice bath and an ultrasonic probe was inserted for two-stage ultrasonic treatment. The ultrasonic frequency was 20 kHz, and a pulse mode was used with pulse parameters of 3 s on and 3 s off. The ultrasonic power of the first stage was 520 W, and the treatment time was 5 min; the ultrasonic power of the second stage was 180 W, and the treatment time was 5 min. During the ultrasonic treatment, the system temperature was controlled to not exceed 30 ℃ by using an ice bath. After the treatment, ultrasonic treatment solution 5 was obtained.

[0053] (3) Same as step (3) in Example 1.

[0054] (4) Same as step (4) in Example 1, to obtain modified yeast protein powder 5.

[0055] The in vitro simulated gastrointestinal digestibility results of modified yeast protein powder 5 are shown in Table 1.

[0056] Comparative Example 6 A method for preparing yeast protein powder, which differs from Example 1 in that the order of enzyme addition is adjusted, while the other conditions are the same as in Example 1, includes the following steps: (1) Same as step (1) in Example 1.

[0057] (2) Same as step (2) in Example 1, after the treatment is completed, ultrasonic treatment liquid 6 is obtained.

[0058] (3) Adjust the pH of the ultrasonic treatment solution 6 to 5.0, add 0.48 g of β-glucanase (0.8% of yeast protein content), and hydrolyze at 55 °C for 5 h; then adjust the pH of the hydrolysis system to 8.0, add 0.18 g of glutaminase (0.3% of yeast protein content), and hydrolyze at 55 °C for 4 h. After hydrolysis, inactivate the enzyme by incubating at 95 °C for 15 min to obtain modified yeast protein hydrolysate 6.

[0059] (4) Same as step (4) in Example 1, to obtain modified yeast protein powder 6.

[0060] The in vitro simulated gastrointestinal digestibility results of modified yeast protein powder 6 are shown in Table 1.

[0061] Table 1. Results of in vitro simulated gastrointestinal digestibility of modified yeast protein powder As shown in Table 1, the in vitro simulated gastrointestinal digestive properties of the modified yeast protein powder prepared by the method of the present invention (Examples) and other methods (Comparative Examples) are significantly different. The modified yeast protein powders A to C prepared in Examples 1-3 all exhibited high digestibility in both the gastric and intestinal stages.

[0062] The gastrointestinal digestibility of the modified yeast protein powder in Comparative Example 1 was significantly lower than that in the other examples. This is because Comparative Example 1 did not perform ultrasonic treatment on the yeast protein powder, but instead directly subjected it to dual-enzyme treatment. It can be seen that when the original aggregation state of the yeast protein is strong, it is difficult to fully improve the accessibility of the substrate enzyme by relying solely on dual-enzyme action, ultimately resulting in poor gastrointestinal digestibility.

[0063] Although the gastrointestinal digestibility of modified yeast protein powder 2 was higher than that of modified yeast protein powder 1, it was still significantly lower than that of modified yeast protein powders A-C. Compared with the examples, this method only used single-stage ultrasound combined with dual-enzyme treatment, indicating that the effect of single-stage ultrasound on protein dispersion, depolymerization, and structural regulation is not as sufficient as that of two-stage ultrasound.

[0064] Although Comparative Examples 3 and 4 used the same two-stage ultrasound conditions as Example 1, they were treated with only a single enzyme preparation (β-glucanase or glutaminase), which made it difficult to simultaneously achieve the two objectives of protein conformation regulation and polysaccharide barrier weakening. Therefore, the gastrointestinal digestibility of the modified yeast protein powder was not as good as that of the two-enzyme sequential treatment.

[0065] Comparative Example 5 used the same dual-enzyme treatment conditions as Example 1, but the order of the two-stage ultrasound was adjusted, resulting in the modified yeast protein powder 5 having lower digestibility than Example 1. This indicates that low-intensity ultrasound followed by high-intensity ultrasound is more conducive to the dispersion, depolymerization, and subsequent structural regulation of yeast proteins.

[0066] Comparative Example 6 used the same two-stage ultrasonic conditions as Example 1, but the order of addition of the two enzymes was adjusted, resulting in the lower digestibility of the modified yeast protein powder 6 compared to Example 1. This shows that the synergistic effect of the two enzymes also depends on the reasonable order of addition. Changing the order makes it difficult to fully exert the synergistic effect on protein conformation regulation and polysaccharide barrier weakening.

[0067] Table 2. Solubility results of yeast protein ultrasonic treatment solution Furthermore, as shown in Table 2, there are significant differences in the solubility of yeast protein treatment solutions obtained by different ultrasonic treatment methods. Specifically, the solubility of ultrasonically treated solutions A, B, and C is significantly higher than that of untreated yeast protein dispersion 1 and single-stage ultrasonically treated solution 2. These results indicate that, compared with untreated and single-stage ultrasonic treatment, two-stage ultrasonic treatment is more beneficial for improving the solubility of yeast protein in aqueous systems.

[0068] Figure 1The results show that the peak shape and relative intensity near the amide I and amide II bands in Example 1 changed, indicating that the secondary structure composition and intermolecular non-covalent interactions were adjusted. Compared with the relatively dense β-sheet aggregated state of untreated yeast protein, the two-stage sonication treatment caused the protein molecules to undergo moderate rearrangement after a certain degree of unfolding, thereby forming a structural state that is more conducive to dispersion and subsequent enzymatic digestion.

[0069] Figure 2 This indicates that after ultrasonic treatment, the absolute value of the ζ-potential on the surface of yeast protein particles was significantly reduced, and there were significant differences between the multi-stage ultrasonic treatment group, the single-stage ultrasonic treatment group, and the untreated group. p <0.05). This result indicates that ultrasonic treatment modulates the charge distribution and interfacial state on the surface of yeast protein particles. This change is beneficial for improving the dispersion and hydration capacity of yeast protein particles, thereby promoting the dissolution of yeast proteins in the aqueous phase and providing more favorable conditions for subsequent contact between enzyme molecules and substrates.

[0070] Figure 3 The results showed that the average particle size of yeast protein particles was significantly reduced after ultrasonic treatment, and the average particle size of the samples treated in both stages of ultrasonication was significantly lower than that of the single-stage modified yeast protein and the unmodified yeast protein, with significant differences among the treatment groups. p <0.05). This result indicates that two-stage sonication is more conducive to promoting the deagglomeration and particle refinement of yeast protein aggregates, thereby improving the uniformity of system dispersion and providing more favorable conditions for subsequent full contact between enzyme molecules and yeast protein substrates. The above results show that two-stage sonication can improve the dispersibility and solubility of yeast proteins in terms of protein structure, particle surface state, and particle size. Higher solubility indicates that the substrate has a better dispersion state and higher accessibility in the system, which is conducive to the full contact between glutaminase and β-glucanase and the substrate, thereby improving the efficiency of dual-enzyme treatment and ultimately promoting the improvement of the in vitro simulated gastrointestinal digestive performance of yeast proteins.

[0071] In summary, the superiority of this embodiment over other comparative examples lies in its use of more suitable two-stage ultrasound conditions, combined with sequential treatment by glutaminase and β-glucanase. This results in a more coordinated improvement in the dispersion, aggregation, molecular conformation, and polysaccharide barrier function of yeast proteins. The two-stage ultrasound not only enhances the solubility of the ultrasound treatment solution but, more importantly, provides more favorable substrate conditions for subsequent dual-enzyme treatment. Sequential dual-enzyme treatment further improves protein release and digestibility. Therefore, the two-stage ultrasound combined with dual-enzyme treatment method described in this invention can more effectively improve the in vitro simulated gastrointestinal digestibility of yeast proteins, demonstrating superior technical performance.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing highly digestible yeast protein, characterized in that... Includes the following steps: (1) Disperse yeast protein powder in water and perform the first stage of ultrasonic treatment. The ultrasonic frequency is 15~20 kHz, the ultrasonic power is 100~350 W, and the treatment time is 3~10 min. Then perform the second stage of ultrasonic treatment. The ultrasonic frequency is 15~20 kHz, the ultrasonic power is 400~600 W, and the treatment time is 3~10 min to obtain ultrasonic treatment solution. (2) Adjust the pH of the ultrasonic treatment solution obtained in step (1) to 7.0~8.0, add glutaminase, and hydrolyze at 45~55 ℃ for 4~6 h; then adjust the pH of the hydrolysis system to 4.0~5.0, add β-glucanase, and hydrolyze at 45~55 ℃ for 5~8 h. After the hydrolysis is completed, inactivate the enzyme to obtain a hydrolysate containing yeast protein with high digestibility. In step (2), the amount of glutaminase added is 0.05-0.5% of the mass of the yeast protein powder; In step (2), the amount of β-glucanase added is 0.2~1.0% of the mass of yeast protein powder.

2. The preparation method according to claim 1, characterized in that: The ultrasonic treatment in step (1) is performed in a pulse mode, with pulse parameters of 3-5 seconds on and 3-5 seconds off.

3. The preparation method according to claim 1, characterized in that: The ultrasonic power in the first stage of step (1) is 150~250 W.

4. The preparation method according to claim 1, characterized in that: In step (2), the ultrasonic power in the second stage is 450~550 W.

5. The preparation method according to claim 1, characterized in that: In step (2), the amount of glutaminase added is 0.1 to 0.3% of the mass of yeast protein powder.

6. The preparation method according to claim 1, characterized in that: In step (2), the amount of β-glucanase added is 0.5 to 0.8% of the mass of yeast protein powder.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The enzymatic hydrolysate containing highly digestible yeast protein in step (2) is further concentrated and spray-dried to obtain highly digestible yeast protein powder.

8. The preparation method according to claim 7, characterized in that: The concentration refers to concentrating the material to a dry matter content of 20-25%.

9. A yeast protein with high digestibility, characterized in that: It is prepared by the method described in any one of claims 1 to 8.

10. The application of the highly digestible yeast protein of claim 9 in the preparation of functional foods, fortified foods, and foods for special medical purposes.

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