Enzymolysis process of yeast milk based on surfactant-assisted enzymolysis

Abstract

The invention relates to the technical field of enzymolysis yeast milk, and discloses an enzymolysis process of yeast milk based on surfactant-assisted enzymolysis, which comprises the following steps: adding a surfactant glycerol polyoxyethylene polyoxypropylene ether into autolysis yeast milk, stirring and activating, adding cell wall lytic enzyme, carrying out enzymolysis process, then adding helicase, and carrying out enzymolysis to obtain the yeast milk. And finally, papain is added to carry out the enzymolysis process, so that the yeast milk subjected to enzymolysis is obtained. According to the method, a process strategy of combining an enzymolysis method and a surfactant-assisted enzymolysis method is creatively adopted, the accessibility of a substrate is enhanced by utilizing a surfactant glycerol polyoxyethylene polyoxypropylene ether, the contact efficiency of enzyme and the substrate is further improved, the hydrolysis reaction rate is increased, and the comprehensive production cost is effectively reduced while the production efficiency is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzymatic hydrolysis of yeast milk, in particular to an enzymatic hydrolysis process of yeast milk based on surfactant-assisted enzymatic hydrolysis. BACKGROUND

[0002] With the general improvement of global living standards, consumers' demand for healthy, nutritious and safe food continues to grow, driving the food industry's demand for natural, high-quality functional raw materials to expand. Under this background, yeast extract, with its safety, nutritional richness and natural source, has gradually become an important raw material with the dual functions of flavor enhancement and nutrition supplementation, attracting widespread attention from the industry. Yeast extract, also known as yeast flavor, yeast essence, and yeast extract, is a water-soluble extract prepared from edible yeast through enzymatic hydrolysis, autolysis, and hydrolysis processes. The product forms include powder, paste, and liquid. It is rich in amino acids, small peptides and other soluble components in yeast cells, not only has good flavor enhancement effect and high nutritional value, but also has the advantages of wide source and controllable production cost, and is widely used in the food industry.

[0003] As an important natural flavoring, the core production process of yeast extract is yeast milk enzymatic hydrolysis. Yeast milk enzymatic hydrolysis refers to the use of yeast's own enzyme system to decompose and transform the intracellular components of yeast under specific environmental conditions, ultimately producing yeast extract with rich flavor and comprehensive nutrition. However, the current yeast milk enzymatic hydrolysis process, especially the traditional autolysis method, faces significant technical bottlenecks in actual industrial production. On the one hand, the yeast cell wall structure is dense, and only relying on its own endogenous enzymes cannot completely break the wall, resulting in a large amount of protein, nucleic acid and other nutrients being trapped in the cell and unable to be released, causing the final product yield to be low, and the key flavor indicators such as amino nitrogen and flavor nucleotides to be limited. On the other hand, to solve this problem, the introduction of additional enzymatic hydrolysis method, due to the high cost of enzyme preparation, the incomplete enzymatic hydrolysis caused by the specificity of single enzyme target, and other problems, makes it difficult to balance production efficiency and economic benefit. These factors together restrict the quality improvement and cost control of yeast extract products, and become a common problem that the industry needs to break through.

[0004] With the expanding market demand for yeast extract, the yield and quality problems in its production process have become increasingly prominent. The structure of yeast cell wall has become one of the key bottlenecks restricting the extraction efficiency. The current methods for hydrolyzing yeast cell wall mainly include autolysis and enzymatic hydrolysis: in the autolysis method, the endogenous enzymes of yeast are relied on to decompose the intracellular nutrients, which has the advantages of excellent flavor and low cost, but has problems such as low content of flavor nucleotides, incomplete change of cell wall structure, etc., resulting in insufficient release of intracellular products and low overall yield; in the enzymatic hydrolysis method, the addition of exogenous enzyme preparations can promote the decomposition of cell wall, which has the advantages of convenient production, prevention of bacteria, etc., but also faces challenges such as high cost of enzyme preparations, difficulty in guaranteeing the quality and batch stability of products, etc. Although the existing process has improved the extraction efficiency of nutrients to some extent, the yield, purity and stability of its products are still difficult to meet the growing market demand. These problems not only limit the production efficiency and product consistency of yeast extract, but also restrict its further application in high-value food field. Therefore, developing a high-efficiency and high-quality yeast milk enzymatic hydrolysis process to effectively solve the problem of insufficient enzymatic hydrolysis caused by autolysis and enzyme specificity has become the key to breaking through the current industrial bottleneck, reducing production cost and responding to the market demand for high-quality raw materials, which has important significance for improving the core competitiveness of enterprises and promoting the technological upgrading of the industry. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides an enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis, which solves the problems of incomplete hydrolysis of macromolecules caused by the decrease of endogenous enzyme activity in the autolysis method of yeast milk, low content of flavor nucleotides, high cost of enzyme preparations in the enzymatic hydrolysis method, and enzyme specificity limiting the efficiency of enzymatic hydrolysis, as well as the problems of waste of residual nutrients in yeast fermentation broth, equipment wear caused by separation residues, and difficulty in treating wastewater.

[0006] The technical scheme of the present application: an enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis: (1) Add yeast milk to distilled water and stir to prepare a yeast suspension, add ethyl acetate to promote the rupture of yeast cell wall, and then enhance the subsequent dissolution effect; add citric acid and sodium hydroxide aqueous solution dropwise to adjust the pH of the yeast suspension, and then perform autolysis reaction to obtain autolyzed yeast milk.

[0007] (2) Add surfactant glycerol polyoxyethylene polyoxypropylene ether to the autolyzed yeast milk, stir to activate, add cell wall lyase, perform enzymatic hydrolysis 1 process, then add helicase, perform enzymatic hydrolysis 2 process, and finally add papain, perform enzymatic hydrolysis 3 process to obtain enzymatically hydrolyzed yeast milk.

[0008] Preferably, in (1), the pH of the yeast suspension is adjusted to 4.8-5.2.

[0009] Preferably, the autolysis reaction in (1) is carried out at 27-33℃ for 12-18h.

[0010] Preferably, the mass concentration of glycerol polyoxyethylene polyoxypropylene ether in the autolysing yeast milk in (2) is 0.015-0.045%.

[0011] Preferably, the temperature for stirring and activation is 20-30℃, and the time is 1-5h.

[0012] Preferably, the mass concentration of cell wall lysing enzyme in the autolytic yeast milk in (2) is 1-2%.

[0013] Preferably, in (2), the temperature of the enzymatic hydrolysis process 1 is 40-60℃, the time is 13-17h, and the pH of the autolysed yeast milk is controlled to be 5-7.

[0014] Preferably, the mass concentration of helicase in the autolysed yeast milk in (2) is 0.3-1.1%.

[0015] Preferably, the temperature of the enzymatic hydrolysis process in (2) is 46-62℃, the time is 7-19h, and the pH of the autolysed yeast milk is controlled to be 6-8.

[0016] Preferably, the mass concentration of papain in the autolysed yeast milk in (2) is 1.6-2%.

[0017] Preferably, in (2), the temperature of the enzymatic hydrolysis process 3 is 50-70℃, the time is 8-16h, and the pH of the autolysed yeast milk is controlled to be 6.5-8.5.

[0018] Preferably, the preparation method of yeast milk is as follows: Yeast strains cultivated in a sterile room are first inoculated into a primary seed tank for expansion culture for 15 hours at a temperature of 30.7℃ and a pH of 3.6±0.2. After completion, they are transferred to a secondary seed tank for further expansion culture for 13 hours at a temperature of 30℃ and a pH of 4.5±0.2. Subsequently, they are transferred to a fermenter for fermentation culture at a temperature of 30℃ and a pH of 4.5±0.5. After the culture is completed, the material is centrifuged, and the separated seed liquid is directly transferred to a seed liquid storage tank for later use. Then, the seed liquid is taken from the seed liquid storage tank and inoculated into the fermenter for fermentation. After fermentation is completed, the fermentation broth is removed from the tank, and the resulting fermentation broth is centrifuged again. The supernatant after separation is collected as yeast milk.

[0019] Beneficial Technical Effects: This invention breaks through the core bottlenecks in yeast extract production, namely "insufficient enzymatic hydrolysis, low yield, and unstable quality." It completely solves the problems of incomplete hydrolysis of macromolecules and low content of flavor nucleotides caused by decreased endogenous enzyme activity in autolysis methods, as well as the high cost of enzyme preparations and the limitation of enzyme specificity on hydrolysis efficiency in enzymatic hydrolysis methods. These problems are accompanied by wasted nutrients in yeast fermentation broth, equipment wear from separation residues, and difficult wastewater treatment. This invention aims to establish a "high-efficiency, low-cost, and low-pollution" yeast milk enzymatic hydrolysis process system through a technical path of "screening optimal conditions for single complex proteases → optimizing complex enzyme formulations → synergistic enhancement with nonionic surfactants," combined with response surface methodology. It also develops the core technology of "synergistic cell wall disruption by complex enzymes and surfactants," ultimately achieving the comprehensive goals of improving the quality of yeast extract products while reducing production costs, minimizing environmental pressure, and enhancing product market competitiveness.

[0020] This invention has multiple significances. Technically, through the integrated innovation of synergistic enzymatic hydrolysis with surfactant-assisted technology, it overcomes the technical bottlenecks of traditional autolysis and single enzymatic hydrolysis methods in terms of yeast cell wall disruption efficiency and product yield, establishing a highly efficient and controllable deep enzymatic hydrolysis process system, providing a new technical solution for the industry. Economically, it can significantly increase the amino nitrogen and total nitrogen content of yeast extracts, directly improving product quality and increasing production yield. While optimizing the enzymatic hydrolysis process and reducing production unit consumption, it further enhances product market competitiveness, bringing considerable economic returns to enterprises and achieving the dual benefits of quality improvement and cost reduction. Environmentally, through deep enzymatic hydrolysis technology, it maximizes the degradation of macromolecules within yeast cells, reducing the content of recalcitrant organic matter in fermentation residues and wastewater at the source, effectively reducing the pressure of subsequent environmental treatment and meeting the requirements of green manufacturing and sustainable development. In terms of industrial promotion, it improves the overall technical level of my country's yeast extract industry, promotes the upgrading of the condiment industry towards natural, healthy, and high-quality products, and provides important reference for process innovation in related biomanufacturing fields, having a broad industry-driving effect.

[0021] The experimental content of this invention is as follows: First, using the amino nitrogen content and total nitrogen content of yeast extract as core evaluation indicators, a combination of "single-factor experiments + response surface methodology" was employed to systematically explore the optimal enzymatic hydrolysis conditions for three single complex proteases. The influence of key parameters on the hydrolysis efficiency of each enzyme was investigated, and the optimal process parameters were determined through data fitting and validation, providing precise basic parameter support for subsequent complex enzyme compounding experiments. Second, based on the aforementioned optimal conditions for single proteases, the effects of different addition times of the three complex proteases on the enzymatic hydrolysis efficiency of yeast milk were examined, further optimizing the synergistic effect of the complex enzymes and selecting the compounding scheme that maximizes hydrolysis efficiency. Finally, under the determined optimal compounding conditions, nonionic surfactants were added as enzyme promoters. Using amino nitrogen content as the core indicator, the combined effect of the complex enzymes and nonionic surfactants on improving enzymatic hydrolysis efficiency was examined by comparing the results of the experimental groups with and without surfactants, ultimately forming a highly efficient and economical yeast milk enzymatic hydrolysis process combination.

[0022] This invention, through systematic single-factor experiments, clarified the optimal process conditions for three proteases in a yeast milk hydrolysis system. Specifically, the optimal parameter combination for cell wall lysin was: enzyme dosage 1.5%, pH 6.0, hydrolysis time 15 h, and hydrolysis temperature 50 °C; for helicase, it was: enzyme dosage 0.7%, pH 7.0, hydrolysis time 13 h, and hydrolysis temperature 54 °C; and for papain, it was: enzyme dosage 1.8%, pH 7.5, hydrolysis time 12 h, and hydrolysis temperature 60 °C. Based on this, the response surface methodology was further used to optimize the complex protease process, and the optimal conditions were determined as follows: cell wall lysin: hydrolysis temperature 50℃, hydrolysis pH 6.0, enzyme addition 1.5%; helicase: hydrolysis temperature 54℃, hydrolysis pH 7.0, enzyme addition 0.7%; papain: hydrolysis temperature 60℃, hydrolysis pH 7.5, enzyme addition 1.8%. Under these conditions, the amino nitrogen and total nitrogen contents of cell wall lysin, helicase, and papain were 3.83%±0.03 and 9.64%±0.04, 3.93%±0.03 and 9.78%±0.05, and 4.11%±0.02 and 9.86%±0.03, respectively, indicating that the amino nitrogen content was the highest and the hydrolysis effect was the most significant under these conditions.

[0023] This invention constructs a complex enzymatic hydrolysis system by selecting three different types of complex protease preparations with varying enzymatic hydrolysis characteristics. This effectively overcomes the problem of insufficient enzymatic hydrolysis caused by the catalytic specificity of a single enzyme, broadens the target range of enzymatic hydrolysis, and thus significantly improves the product yield of yeast extract, increases the production of yeast extract, and brings considerable economic benefits to enterprises.

[0024] To further improve enzymatic hydrolysis efficiency, this invention innovatively adopts a process strategy combining "enzymatic hydrolysis + surfactant-assisted enzymatic hydrolysis." The surfactant enhances substrate accessibility, further improving the contact efficiency between the enzyme and substrate, accelerating the hydrolysis reaction rate, and effectively reducing overall production costs while increasing production efficiency. A nonionic surfactant, glycerol polyoxyethylene polyoxypropylene ether, was introduced as an enzyme promoter to investigate its synergistic effect. Results showed that the control group without surfactant had the lowest amino nitrogen and total nitrogen contents. With increasing surfactant addition, the amino nitrogen content gradually increased, reaching a peak at 0.030%, and then stabilized. Finally, under the synergistic effect of the compound enzyme and surfactant, the amino nitrogen content of the enzymatic hydrolysis product increased to 4.57% ± 0.03%, and the total nitrogen content reached 10.22% ± 0.02%, both meeting the preset targets (amino nitrogen ≥ 4.5%, total nitrogen ≥ 10%), effectively verifying the feasibility and advancement of the "compound enzyme + surfactant" synergistic cell-wall breaking technology.

[0025] The enzymatic hydrolysis process in this invention significantly improves product yield and quality, while optimizing enzyme preparation usage strategies and introducing highly efficient enzyme promoters to achieve reasonable control of production costs. This provides a reliable technical path for industrial application and fully achieves the established targets.

[0026] The successful implementation of this invention provides enterprises with an innovative process route to improve the quality and production efficiency of yeast extract products. Through the established synergistic hydrolysis system of "compound enzyme + surfactant," yeast extracts with high amino nitrogen and high total nitrogen content can be stably produced, significantly enhancing the product's market competitiveness in terms of flavor characteristics, nutritional quality, and application performance, and better meeting the food industry's demand for high-quality natural flavoring ingredients.

[0027] At the level of industry technology promotion, the enzymatic hydrolysis process developed by this invention has clear parameters and strong operational controllability, making it easy to implement process transformation and scale-up on existing production facilities. It has broad applicability in the fields of yeast extract and related biomanufacturing. This technology system provides an effective solution to common technical problems that have long existed in the industry, such as insufficient enzymatic hydrolysis and low product yield, and has the potential to become a model for industry technology upgrading, with broad application prospects.

[0028] From a social and environmental benefit perspective, this process maximizes the extraction of nutrients from yeast cells through deep enzymatic hydrolysis, reducing the amount of fermentation residue generated at the source and alleviating the operational load on subsequent environmental treatment systems, thus aligning with the principles of green manufacturing and sustainable development. Simultaneously, a stable supply of high-quality yeast extract will provide excellent raw material support for downstream industries such as condiments, health foods, and specialty feeds, driving the transformation and upgrading of these industries towards higher quality and higher added value. Attached Figure Description

[0029] Figure 1 This study investigates the effect of the amount of cell wall dissolving enzyme added on the hydrolysis efficiency of yeast emulsion.

[0030] Figure 2 This study investigated the effect of helicase addition on the hydrolysis efficiency of yeast emulsion.

[0031] Figure 3 This study investigated the effect of papain addition on the hydrolysis efficiency of yeast emulsion.

[0032] Figure 4 This study investigates the effect of pH on the hydrolysis efficiency of yeast emulsion by cell wall enzymes.

[0033] Figure 5 This study investigates the effect of helicase hydrolysis pH on the hydrolysis efficiency of yeast milk.

[0034] Figure 6 This study investigates the effect of pH on the hydrolysis efficiency of yeast emulsion using papain.

[0035] Figure 7 This study investigates the effect of cell wall enzyme hydrolysis temperature on the hydrolysis efficiency of yeast emulsion.

[0036] Figure 8 This study investigates the effect of helicase hydrolysis temperature on the hydrolysis efficiency of yeast emulsion.

[0037] Figure 9 This study investigates the effect of papain hydrolysis temperature on the hydrolysis efficiency of yeast emulsion.

[0038] Figure 10 This study investigates the effect of cell wall enzyme hydrolysis time on the hydrolysis efficiency of yeast emulsion.

[0039] Figure 11 This study investigates the effect of helicase hydrolysis time on the hydrolysis efficiency of yeast emulsion.

[0040] Figure 12 This study investigates the effect of papain hydrolysis time on the hydrolysis efficiency of yeast emulsion.

[0041] Figure 13 It is the effect of the interaction of different factors of cell wall enzymes on the amino nitrogen content.

[0042] Figure 14 It is the effect of the interaction of different factors of cell wall enzymes on the total nitrogen content.

[0043] Figure 15 This describes the effect of the interaction of different factors on the amino nitrogen content of helicase.

[0044] Figure 16 This describes the effect of the interaction of different factors on the total nitrogen content of helicase.

[0045] Figure 17 This study investigates the effect of the interaction of different factors on the amino nitrogen content of papain.

[0046] Figure 18 This study investigates the effect of the interaction of different factors on the total nitrogen content of papain.

[0047] Figure 19 This study investigates the effect of surfactant addition on the hydrolysis efficiency of yeast milk. Detailed Implementation

[0048] The following is the model number of glycerol polyoxyethylene polyoxypropylene ether: DF 103T.

[0049] Example 1: Yeast strains cultured in a sterile room were first inoculated into a primary seed tank for expansion culture for 15 hours at 30.7°C and pH 3.6. After expansion, they were transferred to a secondary seed tank for further expansion, cultured for 13 hours at 30°C and pH 4.5. Subsequently, they were transferred to a fermenter for fermentation culture at 30°C and pH 4.5. After cultivation, the material was centrifuged, and the separated seed liquid was directly transferred to a seed liquid storage tank for later use. The seed liquid was then taken from the storage tank and inoculated into the fermenter for fermentation. After fermentation, the fermentation broth was removed from the fermenter, and the resulting fermentation broth was centrifuged again. The supernatant was collected as yeast milk.

[0050] Yeast milk was added to distilled water and stirred to prepare a yeast suspension with a mass concentration of 13%. Ethyl acetate was added to promote the rupture of yeast cell walls, thereby enhancing the subsequent dissolution effect. Citric acid and sodium hydroxide aqueous solution were added dropwise to adjust the pH of the yeast suspension to 5. The autolysis reaction was carried out at 30℃ for 12 hours to obtain autolyzed yeast milk.

[0051] Compound enzyme hydrolysis test, single-factor experiment on enzyme dosage: The autolyzed yeast milk was subjected to enzymatic hydrolysis with cell wall lysin, helicase, and papain, respectively. Optimal conditions were screened using single-factor experiments. The optimal enzyme dosage for each enzyme was determined using the amino nitrogen content and total nitrogen content of the product as evaluation indicators. Specifically, for cell wall lysin, the hydrolysis temperature was fixed at 50℃, pH 6.0, and the time at 15 h, with enzyme dosages of 1.00%, 1.30%, 1.50%, 1.70%, and 2.00%; for helicase, the hydrolysis temperature was fixed at 54℃, pH 7.0, and the time at 13 h, with enzyme dosages of 0.30%, 0.50%, 0.70%, 0.90%, and 1.10%; and for papain, the hydrolysis temperature was fixed at 60℃, pH 7.5, and the time at 12 h, with enzyme dosages of 1.60%, 1.70%, 1.80%, 1.90%, and 2.00%.

[0052] Enzyme pH Single Factor Assay: Single-factor experiments were conducted with the enzyme dosage, hydrolysis temperature, time, and pH of the three proteases fixed as variables to screen for optimal conditions. The amino nitrogen content and total nitrogen content of the products were used as evaluation indicators. Specifically, the conditions were: cell wall lysin: fixed enzyme dosage 1.5%, hydrolysis temperature 50℃, time 15h, pH 5.0, 5.5, 6.0, 6.5, 7.0; helicase: fixed enzyme dosage 0.7%, hydrolysis temperature 54℃, time 13h, pH 6.0, 6.5, 7.0, 7.5, 8.0; papain: fixed enzyme dosage 1.8%, hydrolysis temperature 60℃, time 12h, pH 6.5, 7.0, 7.5, 8.0, 8.5.

[0053] Single-factor experiment on enzymatic hydrolysis temperature: The enzyme dosage, hydrolysis pH, and time of three proteases were fixed. Single-factor experiments were conducted with hydrolysis temperature as the variable, and the optimal conditions were screened using the amino nitrogen content and total nitrogen content of the products as evaluation indicators. Specifically, the conditions were set as follows: cell wall lysin: fixed enzyme dosage 1.5%, pH 6.0, hydrolysis time 15 h, temperatures 40℃, 45℃, 50℃, 55℃, and 60℃; helicase: fixed enzyme dosage 0.7%, pH 7.0, hydrolysis time 13 h, temperatures 46℃, 50℃, 54℃, 58℃, and 62℃; papain: fixed enzyme dosage 1.8%, pH 7.5, hydrolysis time 12 h, temperatures 50℃, 55℃, 60℃, 65℃, and 70℃.

[0054] Single-factor experiment on enzymatic hydrolysis time: The enzyme dosage, hydrolysis temperature, and pH of three proteases were fixed, and single-factor experiments were conducted with hydrolysis time as the variable. The optimal conditions were screened using the amino nitrogen content and total nitrogen content of the products as evaluation indicators. Specifically, the conditions were set as follows: cell wall lysin: fixed enzyme dosage 1.5%, temperature 50℃, pH 6.5, hydrolysis time 13h, 14h, 15h, 16h, 17h; helicase: fixed enzyme dosage 0.7%, temperature 54℃, pH 7.0, hydrolysis time 7h, 10h, 13h, 16h, 19h; papain: fixed enzyme dosage 1.8%, temperature 60℃, pH 7.5, hydrolysis time 8h, 10h, 12h, 14h, 16h.

[0055] Response surface methodology for process optimization: Single-factor experiments showed that the hydrolysis time had a relatively small impact on the hydrolysis effect; therefore, the hydrolysis time factor was excluded in subsequent single-factor experiments. Based on the single-factor analysis, response surface methodology (RSM) was used to optimize the effects of enzyme addition (%), hydrolysis pH, and hydrolysis temperature (°C) on the hydrolysis effect of yeast milk. Seventeen schemes were designed for three variables and three levels according to the Box-Behnken (BBD) experimental method, with each variable located at three equidistant levels (-1, 0, +1). The amino nitrogen content and total nitrogen content of the product were used as response values, as shown in Tables 1, 2, and 3 below.

[0056] Table 1. Optimization factors and their coding for cell wall lysin response surface methodology.

[0057] Table 2. Levels and codes of optimization factors for helicase response surface methodology.

[0058] Table 3. Optimization factors and coding for papain response surface methodology

[0059] Experiment on the effect of compound enzyme hydrolysis: Based on the amino nitrogen content gradient values ​​and significance analysis results obtained from the individual verification experiments of the three proteases, the optimal conditions of the single enzymes were first fixed, namely enzyme addition amount, temperature and pH value. Then, by setting different compound enzyme addition times, the effect of the compound enzyme system on the hydrolysis effect of yeast milk was investigated.

[0060] The effect of adding enzyme promoters on the enzymatic hydrolysis of yeast milk: Under the established optimal reaction conditions of the compound enzyme, a nonionic surfactant was added, and parallel experiments were conducted with different addition amounts and times. First, the addition amount was set at 0.015%, and the addition time was set at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h; the optimal addition time was screened using amino nitrogen content and total nitrogen content as evaluation indicators. Second, based on the optimal addition time, addition amounts of 0.015%, 0.020%, 0.025%, 0.030%, 0.035%, 0.040%, and 0.045% were set, and the effects of enzyme promoter addition on the hydrolysis efficiency of yeast milk were investigated using amino nitrogen and total nitrogen content as indicators.

[0061] Data analysis: Data processing and analysis were performed using Origin 2024 and SPSS 27.0 software. Numerical values ​​are expressed as mean ± standard error. One-way ANOVA and LSD test were used for inter-group comparisons. P < 0.05 indicated statistical significance, and P < 0.01 indicated significant difference.

[0062] The effect of enzyme dosage on the hydrolysis efficiency of yeast emulsion is shown in [reference needed]. Figures 1-3 As can be seen, the amino nitrogen content is represented by a line graph, and the total nitrogen content is represented by a bar graph, and the same applies below. It can be seen that with the increase of enzyme addition, the contents of both nitrogen types show a trend of first increasing and then decreasing. The optimal addition amounts for the three proteases are: 0.7% for cell wall lysin, 1.5% for helicase, and 1.8% for papain. Under these conditions, the amino nitrogen and total nitrogen contents for the cell wall lysin group are 3.74%±0.04 and 9.55%±0.03, respectively; for the helicase group, 3.85%±0.02 and 9.71%±0.03; and for the papain group, 3.98%±0.04 and 9.75%±0.04. This may be due to differences in the optimal reaction conditions and substrate affinity of different enzymes, resulting in different addition amounts required to achieve maximum hydrolysis efficiency. This indicates that the peak of total nitrogen release is not completely synchronized with that of amino nitrogen under the action of different enzymes, which may be related to the cleavage sites and extent of peptide bonds in protein molecules by different enzymes. Among the three enzymes, papain exhibited significantly better overall hydrolytic performance than cell wall lysin and helicase. This may be attributed to its broader substrate specificity and synergistic enzymatic cleavage ability, enabling it to more efficiently break down proteins into smaller nitrogen-containing molecules. Therefore, considering both amino nitrogen content and total nitrogen content, the optimal hydrolysis conditions were selected for the addition of cell wall lysin, helicase, and papain at concentrations of 0.7%, 1.5%, and 1.8%, respectively.

[0063] The effect of enzymatic hydrolysis pH on the hydrolysis efficiency of yeast milk is shown in [reference needed]. Figures 4-6 With increasing pH, both amino nitrogen and total nitrogen contents showed a trend of first increasing and then decreasing. The optimal pH for the action of cell wall lysin, helicase, and papain were 6.0, 7.0, and 7.5, respectively. Under these conditions, the amino nitrogen and total nitrogen contents of the cell wall lysin group were 3.76%±0.02 and 9.57%±0.02, respectively; those of the helicase group were 3.87%±0.04 and 9.74%±0.04, respectively; and those of the papain group were 4.02%±0.04 and 9.79%±0.03, respectively. This may be because different enzymes have the highest activity within their optimal pH range, enabling them to more effectively catalyze the hydrolysis of proteins into small amino acids and short peptides. This pH range is conducive to the full dissolution and enzymatic hydrolysis of proteins; excessively low or high pH may cause changes in enzyme conformation or decrease substrate solubility, thus affecting the total nitrogen release effect. Among the three enzymes, papain exhibited the best overall hydrolytic effect, which may be related to its broader enzyme composition and synergistic catalytic ability, enabling it to maintain high protein degradation efficiency under different pH conditions. Therefore, considering both amino nitrogen content and total nitrogen content, pH values ​​of 6.0, 7.0, and 7.5 for cell wall lysin, helicase, and papain were selected as the optimal hydrolysis conditions.

[0064] The effect of enzymatic hydrolysis temperature on the hydrolysis efficiency of yeast milk is shown in [reference needed]. Figures 7-9 It can be seen that with the increase of enzymatic hydrolysis temperature, both the amino nitrogen content and the total nitrogen content show a trend of first increasing and then decreasing. Specifically, the optimal enzymatic hydrolysis temperatures for cell wall lysin, helicase, and papain are 50℃, 54℃, and 60℃, respectively, with corresponding amino nitrogen and total nitrogen contents of 3.80%±0.03 and 9.60%±0.03, 3.90%±0.04 and 9.76%±0.02, and 4.08%±0.03 and 9.82%±0.02. This may be because within the suitable temperature range, increasing the temperature helps to increase the collision frequency between enzyme molecules and substrates and accelerate the reaction rate; however, when the temperature exceeds a certain threshold, the enzyme protein gradually undergoes thermal denaturation, leading to the destruction of its spatial structure and a decrease in catalytic ability, thereby causing a decrease in the content of hydrolysis products. Further increases in temperature may lead to the loss of some nitrogenous substances due to thermal accumulation or precipitation, thus affecting the release and determination of total nitrogen. Among the three enzymes, papain exhibited the best overall hydrolytic effect, which may be related to its more comprehensive enzyme composition and higher thermal stability, enabling it to decompose protein substrates more persistently and efficiently under the same temperature conditions. Therefore, considering both amino nitrogen content and total nitrogen content, the optimal hydrolysis temperatures for cell wall lysin, helicase, and papain were selected as 50℃, 54℃, and 60℃, respectively.

[0065] Effect of enzymatic hydrolysis time on the hydrolysis efficiency of yeast milk: see Figures 10-12 As can be seen, with the increase of enzymatic hydrolysis time, both amino nitrogen content and total nitrogen content showed a trend of first increasing and then decreasing. Specifically, the optimal enzymatic hydrolysis times for amino nitrogen content and total nitrogen content under the action of cell wall lysin, helicase, and papain were 15 h, 13 h, and 12 h, respectively, corresponding to the highest contents of 3.80%±0.04 and 9.61%±0.03, 3.91%±0.03 and 9.76%±0.02, and 4.09%±0.03 and 9.83%±0.02, respectively. This may be because within the optimal time, the enzyme and substrate fully interact, promoting the continuous degradation of proteins into small peptides and amino acids; however, with further extension of time, the hydrolysis rate may slow down or even increase due to product accumulation inhibition, decreased enzyme activity, or substrate depletion, thus causing a decrease in amino nitrogen content. Within this time range, protein dissolution and decomposition are relatively complete; beyond the optimal time, the total nitrogen value may decrease due to decreased system stability or the re-aggregation and precipitation of some nitrogen-containing substances. Among the three enzymes, papain exhibited the best overall hydrolytic effect, which may be related to the synergistic catalysis of its multiple enzyme components, enabling it to decompose different peptide bonds more efficiently in a shorter time, thereby increasing the release rate of amino nitrogen and total nitrogen. Therefore, considering both amino nitrogen content and total nitrogen content, the optimal hydrolysis conditions were selected for cell wall lysin, helicase, and papain, with hydrolysis times of 15 h, 13 h, and 12 h, respectively.

[0066] Response surface methodology analysis of cell wall lysin: The extraction conditions of cell wall lysin were optimized using response surface methodology. Table 4 shows the amino nitrogen content and total nitrogen content in the product under the conditions of enzyme addition (%), hydrolysis pH, and hydrolysis temperature (°C) designed according to the response surface methodology.

[0067] Table 4 Results of the Three-Level Factorial Design

[0068] Using Design-Expert 13 software, analysis of variance and quadratic polynomial regression were performed on the enzymatic hydrolysis dosage (%), hydrolysis pH, hydrolysis temperature (°C), and amino nitrogen content. Table 5 shows that the model has an F-value of 39.97 and a P-value of 3.40e. -5 The value was <0.0001, indicating high significance. In the lack-of-fit analysis, the F-value was 0.32 and the P-value was 0.8122 (>0.05), indicating that the lack-of-fit term was not significant and the experimental error was small, indicating a good model fit. The model's coefficient of determination R² = 0.9809, and the corrected R²... 2 =0.9564, indicating a high goodness of fit and extremely strong correlation and reliability between the experimental and predicted values. Furthermore, factors A and B, as well as the quadratic term A... 2 B 2 C 2 All three factors had a significant impact on amino nitrogen content. As shown by FA=45.76, FB=43.53, and FC=2.79, the order of influence of the three factors on amino nitrogen content is: A>B>C.

[0069] Using Design-Expert 13 software, multiple linear regression and binomial fitting were performed, resulting in the quadratic multinomial regression equation: Amino nitrogen content (%) = 3.79 - 0.1012A - 0.0988B - 0.025C + 0.0025AB - 0.2343A 2 -0.2143B 2 -0.0617C 2 In summary, the relationships between variables established by this response surface methodology are reliable and can be used for the prediction and optimization of amino nitrogen content.

[0070] Table 5. Results of ANOVA on amino nitrogen content. , In the table above, * indicates significant (P<0.05), and ** indicates highly significant (P<0.01).

[0071] Using Box-Behnken design and response surface methodology, response surfaces and contour plots of different factors on amino nitrogen content were obtained. The slope of the three-dimensional response surface can intuitively reflect the strength of the influence of each factor on amino nitrogen content, while the shape and density of the contour lines are used to determine the significance of the interaction between factors. An ellipse indicates a significant interaction between two factors, while a circle indicates an insignificant interaction. The denser the contour lines, the more significant the influence, and the steeper the surface.

[0072] The effects of interactions of different factors on amino nitrogen content are shown in [reference needed]. Figure 13 The response surface methodology (RSM) results show that the interaction between enzyme dosage and enzymatic hydrolysis temperature exhibits a significant slope, with its contour lines clearly elliptical, indicating a highly significant interaction. Similarly, the response surface slope for the interaction between enzyme dosage and enzymatic hydrolysis pH is also relatively pronounced, with elliptical contour lines, indicating a significant interaction; while the contour lines for the interaction between enzymatic hydrolysis pH and enzymatic hydrolysis temperature are nearly circular, indicating a weaker interaction.

[0073] Comprehensive analysis revealed that the influence of various factors on amino nitrogen content was ranked as follows: enzymatic hydrolysis temperature ≈ pH value > enzyme dosage. Response surface methodology optimization yielded the optimal process conditions: enzymatic hydrolysis temperature 49.57℃, enzymatic hydrolysis pH 5.95, and enzyme dosage 1.46%. Three parallel validation experiments were conducted under these conditions, and the amino nitrogen content was measured to be 3.83% ± 0.06%. The results indicate that the process conditions determined by response surface methodology are accurate and reliable, and can be used for practical optimization applications.

[0074] Design-Expert 13 software was used to perform analysis of variance and quadratic regression fitting on the relationship between enzyme dosage, pH value, enzymatic hydrolysis temperature, and total nitrogen content. Table 6 shows that the model F=38.03, P=4.02e -5 The value <0.0001 indicates that the model is highly significant. The F-value for the lack-of-fit term is 0.13, and the P-value is 0.9380 (>0.05), indicating that the lack of fit is not significant, the experimental error is small, the model fits well, and the prediction accuracy is high. The model's coefficient of determination R² = 0.9800, and the corrected R² = 0.9542, indicating that the experimental value and the predicted value are highly correlated, and the model is reliable. Among the factors investigated, each factor and the quadratic term have a significant impact on the total nitrogen content. Based on the F-values ​​FA = 40.36, FB = 30.31, and FC = 6.46, the order of influence of the factors is A > B > C. The quadratic multinomial regression equation is: Total nitrogen content (%) = 9.600 - 0.0937A - 0.0812B - 0.0375C + 0.0075AB + 0.005AC - 0.02BC - 0.2518A 2 -0.1768B 2 -0.0742C 2In summary, response surface methodology (RSM) reliably demonstrates the influence of enzyme dosage, pH, and temperature on total nitrogen content.

[0075] Table 6. Results of ANOVA for Total Nitrogen Content , In the table above, * indicates significant (P<0.05), and ** indicates highly significant (P<0.01).

[0076] from Figure 14 The response surface methodology (RSM) results show that the quadratic term of the response surface for enzymatic hydrolysis temperature has the steepest slope, indicating that this factor has the greatest impact on total nitrogen content. The interaction between enzyme dosage and enzymatic hydrolysis temperature exhibits a significant slope on the response surface, with its contour lines clearly elliptical, indicating a highly significant interaction. Similarly, the response surface slopes for the interactions between enzyme dosage and enzymatic hydrolysis pH, as well as between enzymatic hydrolysis pH and enzymatic hydrolysis temperature, are also quite pronounced, with contour lines all being elliptical, indicating significant interactions.

[0077] Comprehensive analysis showed that the influence of each factor on the total nitrogen content was ranked as follows: enzymatic hydrolysis temperature > pH value > enzyme dosage. Response surface methodology optimization yielded the optimal process conditions: enzymatic hydrolysis temperature 49.59℃, enzymatic hydrolysis pH 5.96, and enzyme dosage 1.46%. Three parallel validation experiments were conducted under these conditions, and the total nitrogen content was measured to be 9.64% ± 0.04%. The results indicate that the process conditions determined by the response surface methodology are accurate and reliable, and can be used for practical optimization applications.

[0078] Helicase Response Surface Methodology Analysis: Response surface methodology was used to optimize the extraction conditions of the helicase. Table 7 shows the amino nitrogen content and total nitrogen content in the product under the conditions of enzyme addition (%), hydrolysis pH, and hydrolysis temperature (°C) designed according to the response surface methodology.

[0079] Table 7 Results of the Three-Level Factorial Design , In the table above, * indicates significant (P<0.05), and ** indicates highly significant (P<0.01).

[0080] The effects of enzyme dosage, pH value, enzymatic hydrolysis temperature, and amino nitrogen content on the model were analyzed using Design-Expert 13 software, followed by analysis of variance and quadratic polynomial regression fitting. Table 8 shows the results: the F-value is 178.08, and the P-value is 2.00e. -7 The value (<0.0001) indicates high significance; the F-value and P-value in the lack-of-fit analysis are 0.06 and 0.9792 (>0.05), respectively, indicating that the lack-of-fit term is not significant and the experimental error is small, indicating good model fit. The model's coefficient of determination R0 is... 2 =0.9957, corrected R 2=0.9901, indicating a high goodness of fit and extremely strong correlation and reliability between the experimental and predicted values. Furthermore, factors A, B, and C, the interaction term AB, and the quadratic term A... 2 B 2 C 2 All four factors had a significant impact on amino nitrogen content. Based on FA=225.4, FB=30.47, and FC=35.54, the order of influence of the four factors on amino nitrogen content is: A>C>B. The specific quadratic polynomial regression equation is: Amino nitrogen content (%) = 3.91 - 0.085A - 0.0313B - 0.0337C + 0.0825AB + 0.0025AC + 0.005BC - 0.213A 2 -0.1355B 2 -0.0605C 2 In summary, the relationships between variables established by this response surface methodology are reliable and can be used for the prediction and optimization of amino nitrogen content.

[0081] Table 8. Results of ANOVA on amino nitrogen content. , In the table above, * indicates significant (P<0.05), and ** indicates highly significant (P<0.01).

[0082] from Figure 15 It can be seen that the response surface curves for enzyme dosage and hydrolysis temperature have the steepest slope, indicating that these two factors have the greatest impact on amino nitrogen content. Furthermore, the contour lines are distinctly elliptical, confirming a highly significant interaction between the two. Similarly, the response surfaces for enzyme dosage and hydrolysis pH, as well as the hydrolysis pH and hydrolysis temperature, also exhibit significant slopes and elliptical contour lines, indicating a significant interaction.

[0083] Comprehensive analysis revealed that the influence of various factors on amino nitrogen content was ranked as follows: enzymatic hydrolysis temperature > enzymatic hydrolysis pH > enzyme dosage. Response surface methodology optimization yielded the optimal process conditions: enzymatic hydrolysis temperature 53.52℃, enzymatic hydrolysis pH 6.96, and enzyme dosage 0.64%. Three parallel validation experiments were conducted under these conditions, and the amino nitrogen content was measured to be 3.94% ± 0.03%. The results indicate that the process conditions determined by response surface methodology are accurate and reliable, and can be used for practical optimization applications.

[0084] As shown in Table 9, the model's F-value is 113.99 and the P-value is 9.37e. -7 <0.0001 indicates that the model is highly significant. The F-value for the lack of fit is 0.17, and P = 0.9109 > 0.05, indicating that the lack of fit is not significant, the experimental error is small, the model fits well, and the prediction accuracy is high. The model's coefficient of determination R0 2 The adjusted R is 0.9932. 2The correlation coefficient is 0.9845, indicating a high correlation between the experimental and predicted values, and the model is reliable. Among the factors examined, A, B, C, and A... 2 B 2 C 2 All three factors had a significant impact on total nitrogen content. Based on FA=153.29, FB=5.98, and FC=14.6, the order of influence of the three factors on total nitrogen content is: A>C>B. The quadratic polynomial regression equation is: Total nitrogen content (%) = 9.75 - 0.1012A - 0.02B - 0.0313C + 0.015AB - 0.0125AC + 0.02BC - 0.2658A 2 -0.1583B 2 -0.0607C 2 In summary, response surface methodology (RSM) reliably demonstrates the influence of enzyme dosage, pH, and temperature on total nitrogen content.

[0085] Table 9. Results of ANOVA for Total Nitrogen Content

[0086] In the table above, * indicates significant (P<0.05), and ** indicates highly significant (P<0.01).

[0087] from Figure 16 It is evident that the response surface methodology (RSM) curves for enzyme dosage and hydrolysis temperature exhibit the steepest slope, indicating a significant impact of these two factors on total nitrogen content. Furthermore, the contour lines are distinctly elliptical, confirming a highly significant interaction between the two factors. Similarly, the RSM curves for enzyme dosage and hydrolysis pH, and for hydrolysis pH and hydrolysis temperature, also show a certain slope, with elliptical contour lines, indicating a significant interaction. In summary, the order of influence of each factor on total nitrogen content is: hydrolysis temperature > hydrolysis pH > enzyme dosage. Response surface methodology optimization yielded the optimal process conditions as follows: hydrolysis temperature 53.59℃, hydrolysis pH 6.97, and enzyme dosage 0.65%. Three parallel verification experiments were conducted under these conditions, yielding a total nitrogen content of 9.78% ± 0.04%. The results demonstrate that the process conditions determined by the response surface methodology are accurate and reliable, and can be used for practical optimization applications.

[0088] Response surface methodology analysis of papain: The extraction conditions of papain were optimized using response surface methodology. Table 10 shows the amino nitrogen content (%) and total nitrogen content (%) of the product under the conditions of enzyme addition (%), hydrolysis pH, and hydrolysis temperature (°C) according to the response surface methodology.

[0089] Table 10 Results of the Three-Level Factorial Design , In the table above, * indicates significant (P<0.05), and ** indicates highly significant (P<0.01).

[0090] Analysis of variance and quadratic polynomial regression were performed using Design-Expert 13 software to study the effects of enzyme dosage, hydrolysis pH, hydrolysis temperature, and amino nitrogen content. Table 11 shows the results: the F-value was 65.42, and the P-value was 6.34. -6 <0.0001, indicating high significance; in the lack-of-fit analysis, the F-value was 0.48, P = 0.7110 > 0.05, indicating that the lack-of-fit term was not significant and the experimental error was small, indicating good model fit. Model coefficient of determination R0 2 =0.9883, adjusted R 2 The coefficient of performance (COP) is 0.9731, indicating a high goodness of fit and extremely strong correlation and reliability between the experimental and predicted values. Furthermore, each factor, its interaction term, and its quadratic term all have a significant impact on the total nitrogen content. From FA=56.75, FB=53.88, and FC=13.47, it can be seen that the order of influence of the three factors on the total nitrogen content is: A>B>C. The quadratic multinomial regression equation is: Amino nitrogen content (%) = 4.11 - 0.0487A - 0.0475B - 0.0237C + 0.0275AB + 0.0100AC + 0.0175BC - 0.148A 2 -0.1005B 2 -0.033C 2 In summary, the relationships between variables established by this response surface methodology are reliable and can be used for the prediction and optimization of amino nitrogen content.

[0091] Table 11 Results of ANOVA for Amino Nitrogen Content , In the table above, * indicates significant (P<0.05), and ** indicates highly significant (P<0.01).

[0092] from Figure 17 It can be concluded that the response surface slope of enzyme dosage versus enzymatic hydrolysis temperature is the steepest, indicating that both have the most significant impact on amino nitrogen content. Furthermore, the contour lines are distinctly elliptical, suggesting a highly significant interaction between the two. Similarly, the response surfaces of enzyme dosage versus enzymatic hydrolysis pH and enzymatic hydrolysis pH versus enzymatic hydrolysis temperature also exhibit a certain slope, with elliptical contour lines, indicating a significant interaction.

[0093] Comprehensive analysis showed that the influence of each factor on the amino nitrogen content was in the following order: enzymatic hydrolysis temperature > enzymatic hydrolysis pH > enzyme dosage. Response surface methodology optimization yielded the optimal process conditions: enzymatic hydrolysis temperature 59.64℃, enzymatic hydrolysis pH 7.45, and enzyme dosage 1.78%. Three parallel validation experiments were conducted under these conditions, and the amino nitrogen content was measured to be 4.12% ± 0.05%. The results indicate that the process conditions determined by response surface methodology are accurate and reliable, and can be used for practical optimization applications.

[0094] Design-Expert 13 software was used to perform analysis of variance and quadratic regression fitting on the relationship between enzyme dosage, hydrolysis pH, hydrolysis temperature, and total nitrogen content. Table 12 shows that the model F-value was 86.31 and the P-value was 2.45e. -6 The value <0.0001 indicates that the model is highly significant. The F-value for the lack-of-fit term is 0.6818, and P = 0.6079 > 0.05, indicating that the lack of fit is not significant, the experimental error is small, the model fits well, and the prediction accuracy is high. The model's coefficient of determination R0 2 The adjusted R is 0.9911. 2 The correlation coefficient is 0.9796, indicating a high correlation between the experimental and predicted values, and the model is reliable. Among the factors examined, A, B, C, and A... 2 B 2 C 2 The effect on amino nitrogen content was extremely significant. The quadratic polynomial regression equation is: Total nitrogen content (%) = 9.86 - 0.05A - 0.0487B + 0.0213C + 0.0175AB + 0.0025AC + 0.01BC - 0.1645A 2 -0.097B 2 -0.047C 2 In summary, response surface methodology (RSM) confirms the reliable relationship between enzyme dosage, hydrolysis pH, and hydrolysis temperature on total nitrogen content.

[0095] Table 12 Results of ANOVA for Total Nitrogen Content , In the table above, * indicates significant (P<0.05), and ** indicates highly significant (P<0.01).

[0096] from Figure 18 It can be seen that the response surface curves for pH versus temperature, pH versus enzyme dosage, and enzyme dosage versus temperature all exhibit significant slopes, indicating that these factors have a large impact on total nitrogen content. Furthermore, the contour lines show a distinct elliptical shape, confirming a highly significant interaction between the factors. Comprehensive analysis shows that the order of influence of each factor on total nitrogen content is: hydrolysis temperature > hydrolysis pH > enzyme dosage. Response surface optimization yielded the optimal process conditions: hydrolysis temperature 59.64℃, hydrolysis pH 7.45, and enzyme dosage 1.78%. Three parallel verification experiments were conducted under these conditions, and the total nitrogen content was measured to be 9.87% ± 0.05%. The results demonstrate that the process conditions determined by the response surface methodology are accurate and reliable, and can be used for practical optimization applications.

[0097] To facilitate practical operation, a pilot-scale test was conducted based on the results of the response surface methodology to further narrow down the range of enzymatic hydrolysis conditions. The hydrolysis conditions for cell wall lysin were modified as follows: hydrolysis temperature 50℃, hydrolysis pH 6.0, and enzyme dosage 1.5%; the hydrolysis conditions for helicase were modified as follows: hydrolysis temperature 54℃, hydrolysis pH 7.0, and enzyme dosage 0.7%; and the hydrolysis conditions for papain were modified as follows: hydrolysis temperature 60℃, hydrolysis pH 7.5, and enzyme dosage 1.8%. Verification showed that under these modified conditions, the amino nitrogen and total nitrogen contents of cell wall lysin, helicase, and papain were 3.83%±0.03 and 9.64%±0.04, 3.93%±0.03 and 9.78%±0.05, and 4.11%±0.02 and 9.86%±0.03, respectively, which were within the expected experimental range.

[0098] Based on the experimental results in Table 13, it can be concluded that the temperature and pH of the cell wall lysin, helicase, and papain gradually increased, as the compound experiment was designed with an initial temperature of 40℃ and pH of 5.0. Subsequently, the pH increased by 0.5 per hour, and the temperature also gradually increased. When 1.5% cell wall lysin was added at pH 6.0 and temperature 50℃, the pH rose to 7.0 and the temperature reached 54℃ after two hours. Then, 0.7% helicase was added, and the pH rose to 7.5 and the temperature rose to 60℃ after one hour. Finally, 1.8% papain was added, and the reaction was carried out for 12 hours. The temperature was then raised to 65℃ and maintained for 30 minutes to inactivate the enzyme. After five parallel experiments, the amino nitrogen content was found to be 4.21% ± 0.04%, and the total nitrogen content was 9.86% ± 0.06%.

[0099] Table 13 Results of Complex Enzyme Combination Analysis

[0100] Analysis of the effect of enzyme promoter addition on yeast milk hydrolysis: Under the optimal reaction conditions of the compound enzyme determined above, nonionic surfactant glycerol polyoxyethylene polyoxypropylene ether was added, and its addition amount was set to 0.015%, 0.020%, 0.025%, 0.030%, 0.035%, 0.040%, and 0.045%. After a certain reaction time, the results of the experimental group without surfactant were compared with the amino nitrogen content as an indicator. The results are shown in Figure 19.

[0101] Depend on Figure 19It can be seen that with the increase of surfactant addition, the amino nitrogen and total nitrogen content in yeast milk hydrolysate showed a trend of first rapidly increasing and then gradually stabilizing. When the addition amount was below 0.030%, the amino nitrogen and total nitrogen content increased significantly with the increase of surfactant addition, reaching peak values ​​of 4.57%±0.03% and 10.22%±0.02% respectively at 0.030%. Further increasing to 0.035%, the amino nitrogen and total nitrogen content did not show a significant increase, but instead showed a decreasing trend. This trend indicates that an appropriate amount of surfactant can effectively promote the enzymatic hydrolysis process. Its mechanism of action may lie in reducing the surface tension of the system and enhancing the contact efficiency between the enzyme and the substrate, thereby increasing the degree of protein hydrolysis. When the addition amount exceeded 0.030%, the promoting effect reached saturation, indicating that there is a reasonable addition threshold.

[0102] Considering both the overall hydrolysis effect and production cost, while adding surfactants helps increase the amino nitrogen content, exceeding 0.030% does not provide further gains and instead leads to increased costs. Therefore, from the perspective of economics in industrial production, a surfactant addition of 0.030% is chosen. This dosage ensures both hydrolysis effectiveness and economic feasibility, meeting the expected targets.

Claims

1. An enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis, characterized in that, The enzymatic hydrolysis process is as follows: (1) Add yeast milk to distilled water and stir to make yeast suspension. Add ethyl acetate to promote the rupture of yeast cell walls and thus enhance the subsequent dissolution effect. Add citric acid and sodium hydroxide aqueous solution to adjust the pH of yeast suspension and then carry out autolysis reaction to obtain autolyzed yeast milk. (2) Add the surfactant glycerol polyoxyethylene polyoxypropylene ether to the autolyzed yeast milk, stir to activate, add cell wall dissolving enzyme to carry out enzymatic hydrolysis 1, then add helicase to carry out enzymatic hydrolysis 2, and finally add papain to carry out enzymatic hydrolysis 3 to obtain enzymatically hydrolyzed yeast milk.

2. The enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis according to claim 1, characterized in that, In step (1), the pH of the yeast suspension is adjusted to 4.8-5.

2.

3. The enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis according to claim 1, characterized in that, The autolysis reaction in (1) is carried out at 27-33℃ for 12-18h.

4. The enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis according to claim 1, characterized in that, In step (2), the mass concentration of glycerol polyoxyethylene polyoxypropylene ether in the autolytic yeast milk is 0.015-0.045%, the stirring activation temperature is 20-30℃, and the time is 1-5h.

5. The enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis according to claim 1, characterized in that, The mass concentration of cell wall dissolving enzyme in the autolytic yeast milk in (2) is 1-2%.

6. The enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis according to claim 1, characterized in that, The temperature of the enzymatic hydrolysis 1 process in (2) is 40-60℃, the time is 13-17h, and the pH of the autolysed yeast milk is controlled at 5-7.

7. The enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis according to claim 1, characterized in that, The mass concentration of helicase in the autolysed yeast milk in (2) is 0.3-1.1%.

8. The enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis according to claim 1, characterized in that, The temperature of the enzymatic hydrolysis process in (2) is 46-62℃, the time is 7-19h, and the pH of the autolysed yeast milk is controlled at 6-8.

9. The enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis according to claim 1, characterized in that, The mass concentration of papain in the autolysed yeast milk in (2) is 1.6-2%.

10. The enzymatic hydrolysis process for yeast milk based on surfactant-assisted enzymatic hydrolysis according to claim 1, characterized in that, The temperature of the enzymatic hydrolysis process in (2) is 50-70℃, the time is 8-16h, and the pH of the autolysed yeast milk is controlled at 6.5-8.5.

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