Yeast protein dairy product and preparation method thereof

Abstract

The invention relates to the technical field of dairy product processing, in particular to a yeast protein dairy product and a preparation method thereof.In the yeast protein preparation process, the quality of the yeast protein dairy product can be remarkably improved through the synergistic effect of key processes such as bi-enzyme directional step-by-step hydrolysis, ultrafiltration desalination concentration, compound bacteria liquid fermentation and an emulsification compound system; the yeast dairy product prepared by the method can be further suitable for products such as high-protein beverages, cheese substitutes and stirred yoghurt, and has the advantages of nutrition retention and texture optimization.

Description

Technical Field

[0001] This invention relates to the field of dairy processing technology, and in particular to a yeast protein dairy product and its preparation method. Background Technology

[0002] Dairy products, as an important component of the human diet, play an irreplaceable role in meeting nutritional needs and promoting growth and development due to their rich content of protein, fat, lactose, vitamins, and minerals. With consumers becoming increasingly health-conscious, higher demands are being placed on the functionality and nutritional diversity of dairy products. Developing new dairy products that are high in protein, easily digestible, and possess outstanding functional properties has become a significant trend in the industry. Protein is one of the core nutrients in dairy products. Traditional dairy products primarily use animal-derived proteins such as cow's milk and goat's milk. While these proteins have high nutritional value, some individuals may experience allergies to animal proteins or lactose intolerance. Furthermore, the amino acid composition of single-origin proteins has limitations, making it difficult to fully meet the human body's balanced requirements for all essential amino acids. Therefore, finding high-quality plant-based or microbial-derived proteins as supplements to optimize the amino acid profile and enhance the nutritional efficacy of dairy products has become a hot research topic in the dairy industry. Yeast protein is a high-quality protein extracted from yeast cells, belonging to the category of microbial protein. As a single-celled fungus, yeast cells are rich in protein. Through specific extraction and purification processes, high-purity yeast protein can be obtained. For example, Chinese invention patent CN119241672B discloses a method for preparing high-purity yeast protein. This method, through steps such as heat treatment of yeast cells, enzymatic cell disruption, and solubilization, combined with fermentation and the use of milk-derived protein peptides, solves the problems of high dietary fiber content and insufficient branched-chain amino acid content in existing yeast protein extraction methods, producing a high-purity yeast protein product with low dietary fiber and high branched-chain amino acid content. From a nutritional perspective, yeast protein has significant advantages: its protein content is typically high, reaching 40%-60% of its dry weight; its amino acid composition is balanced and comprehensive, containing all eight essential amino acids, especially lysine and threonine, which are low in cereal proteins but abundant in yeast protein, effectively compensating for the nutritional deficiencies of plant proteins and meeting the body's balanced amino acid requirements. In terms of functionality, yeast protein not only has a high digestibility and absorption rate, making it suitable for various groups (including infants, the elderly, and other groups with weaker digestive functions), but the yeast cells from which it originates may also contain bioactive substances such as glucan, mannan, and B vitamins. These components work synergistically with yeast protein, giving it additional health benefits such as enhancing immunity, regulating gut microbiota, and promoting nutrient absorption. Furthermore, yeast protein production is characterized by a wide range of raw material sources, a short cultivation cycle, and strong environmental adaptability. Compared to animal protein, its production is less restricted by factors such as land and climate, and it can reduce resource consumption and environmental pollution during the farming process, giving it unique advantages in sustainable development. Therefore, it is considered a highly promising high-quality protein source with broad application prospects in the food and health product industries.

[0003] However, the application of yeast protein in dairy products still faces many challenges. On the one hand, yeast protein itself has a unique flavor, and adding it directly to dairy products can easily lead to unpleasant tastes or odors, affecting consumer acceptance. On the other hand, yeast protein has poor compatibility with milk protein, and precipitation and stratification are prone to occur during processing, damaging the product's stability and textural properties. Furthermore, existing preparation processes suffer from low extraction and purification efficiency of yeast protein, and the technology for compounding it with dairy product base materials is not mature enough, resulting in high production costs and hindering large-scale industrial production. Existing technologies have conducted some research on this topic. For example, Chinese invention patent CN119969527A discloses a method for preparing a protein beverage, which involves adding yeast protein or yeast powder to a liquid, dispersing and hydrating it, and then enzymatically hydrolyzing it with protease and cellohydrolase. This effectively improves the solubility and flavor of yeast protein, providing a technical approach for the application of yeast protein in protein beverages. Hefei University of Technology has developed a yeast protein hydrolysate preparation technology (CN119791255A) that enhances saltiness and promotes digestion and absorption. This technology utilizes flavor protease and neutral protease to enzymatically hydrolyze yeast protein stepwise. The resulting hydrolysate can partially replace sodium chloride and has both blood pressure-lowering and antioxidant functions, expanding the application scenarios of yeast protein in low-sodium functional foods. Angel Yeast Co., Ltd. has developed a yeast protein fermented beverage (CN119498466A), which uses yeast protein, soy protein isolate, and lactic acid bacteria as raw materials for fermentation to prepare a fermented beverage with high total protein content and muscle-building and intestinal health benefits, breaking through the limitations of traditional dairy products and plant protein beverages.

[0004] While the above technologies have solved the application problems of yeast protein to some extent, yeast protein is prone to producing bitter peptides (such as proline oligopeptides) and salty residues (Cl⁻, Na⁺ ions) after enzymatic hydrolysis, resulting in poor product palatability. There is currently a lack of yeast protein dairy product technology that can simultaneously solve the problems of flavor coordination and efficient preparation. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a yeast protein dairy product and its preparation method. During the yeast protein preparation process, the quality of the yeast protein dairy product can be significantly improved through the synergistic effect of key processes such as dual-enzyme directional stepwise hydrolysis, ultrafiltration desalting and concentration, compound bacterial liquid fermentation, and emulsification compounding system. The yeast dairy product prepared by this method is applicable to high-protein beverages, cheese substitutes, and stirred yogurt, possessing advantages in both nutrient retention and texture optimization.

[0006] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution: In a first aspect, this aspect provides a method for preparing a yeast protein dairy product, characterized by comprising the following steps: S1: Dual-enzyme directed stepwise hydrolysis After prehydration, the yeast protein raw material is first subjected to preliminary enzymatic hydrolysis using flavor protease, and then further enzymatic hydrolysis using neutral protease. After the enzymatic hydrolysis is completed, the enzyme is inactivated and cooled to obtain the hydrolysate. S2: Ultrafiltration Desalination and Concentration The enzymatic hydrolysate from step S1 was filtered using an ultrafiltration membrane to retain target proteins with a molecular weight >3 kDa, while removing small peptides and salt ions to obtain the retained product. S3: Compound microbial liquid fermentation Lactose was added to the retentate from step S2 as a fermentation substrate. After sterilization, a compound microbial agent composed of lactic acid bacteria and Pediococcus was inoculated and allowed to ferment statically. The pH at the end of the fermentation was controlled to be 4.0–4.6 to obtain the fermentation substrate. S4: Post-processing and product molding The fermentation substrate is subjected to molding treatment to obtain yeast protein dairy products.

[0007] Furthermore, in the prehydration process of yeast protein raw material in S1, the pH of the system is adjusted to 6.5–7.0 by a pH adjuster, and the ratio of yeast protein raw material to solvent is 1:9-11.

[0008] Furthermore, the enzymatic hydrolysis conditions for the flavor protease are as follows: Preliminary enzymatic hydrolysis: Enzymatic hydrolysis was performed using flavor protease. The hydrolysis conditions were as follows: flavor protease was added at a mass ratio of 2%-4% to substrate, and the reaction system was placed in a constant temperature water bath at 40-45℃ and continuously stirred at a speed of 80-120 rpm for 50-150 minutes. Deep enzymatic hydrolysis: After the initial enzymatic hydrolysis is completed, adjust the pH of the system to 7.0-7.2, raise the temperature to 50-55℃, add neutral protease at an enzyme to substrate mass ratio of 1%-3%, and continue stirring and hydrolyzing for 20-50 minutes; After the reaction is complete, enzyme inactivation treatment is performed; Preferably, the enzyme inactivation treatment is performed by placing the system in a water bath at 80-90°C for 10-15 minutes to inactivate the enzyme, and then cooling it to 37°C.

[0009] Furthermore, the ultrafiltration membrane in S2 is made of polyethersulfone, regenerated cellulose, or ceramic, and operates at a pressure of 0.1–0.2 MPa.

[0010] Furthermore, the compound microbial agent in S3 is composed of lactic acid bacteria and Pediococcus pentosaceus, wherein the lactic acid bacteria are selected from at least one of Lactobacillus casei and Lactobacillus delbrueckii.

[0011] Furthermore, the compound microbial agent is composed of L. casei CICC 6104, L. delbrueckii subsp.lactis CICC 22185 and P. pentosaceus CICC 22227 in a viable count ratio of 4-6:1-3:2-4; The fermentation temperature is 35–39℃, and the fermentation time is 5–8 hours.

[0012] Further, S4 specifically involves: mixing the fermentation base material with the system containing the oil phase and emulsifier and emulsifying it by high-speed shearing, adding a colloidal stabilizer for formulation and then sterilizing, and performing molding treatment according to the product type.

[0013] Furthermore, the oil phase is a vegetable oil, selected from at least one of sunflower seed oil, olive oil, and soybean oil; The emulsifier comprises at least one of sucrose ester, monoglyceride, and monoglyceride succinate; The colloidal stabilizer is a compound of at least two of pectin, carrageenan, and xanthan gum.

[0014] Secondly, this aspect further provides a yeast protein dairy product prepared using the method provided in the first aspect above.

[0015] Furthermore, the yeast protein dairy product is any one of stirred yogurt, cheese substitute, or high-protein beverage.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention significantly optimizes the functionality and quality of yeast protein dairy products through multi-process synergistic innovation. The dual-enzyme targeted stepwise hydrolysis process employs the sequential action of flavor proteases and neutral proteases. The flavor protease preferentially breaks hydrophilic peptide bonds on the protein surface to remove bitter precursors, while the neutral protease further hydrolyzes the exposed hydrophobic regions. Through this synergistic mechanism of "removing bitterness first, then breaking the core," the generation of bitter peptides is effectively reduced, and protein solubility is improved, solving problems such as heavy bitterness and excessive precipitation caused by traditional single-enzyme or simultaneous enzymatic hydrolysis. The ultrafiltration desalting and concentration steps, through precise control of the 3kDa retention threshold and the use of high-performance membrane materials such as polyethersulfone and ceramics, efficiently remove small molecule salt ions and bitter peptides while retaining the activity of the target protein, improving system homogeneity and long-term stability, and avoiding the drawbacks of incomplete desalting or protein loss in traditional processes.

[0017] The compound microbial liquid fermentation system achieves a triple synergistic effect of acid production regulation, flavor synthesis, and off-flavor suppression through a scientific ratio of lactic acid bacteria and Pediococcus. Lactic acid bacteria dominate lactic acid production to maintain system pH stability, while Pediococcus, through esterase activity, decomposes fats and inhibits substance accumulation, while simultaneously promoting the synthesis of acetaldehyde, a characteristic flavor compound, thus imparting a harmonious and natural fermented flavor to the product. The emulsification and compounding technology in the post-processing stage, through the synergistic effect of sucrose esters and monoglycerides, forms a stable interfacial film structure. Combined with the compound use of colloidal stabilizers, this effectively improves oil phase dispersibility and emulsion stability, preventing stratification or flocculation during product storage.

[0018] This process system is applicable to the development of various products such as stirred yogurt, cheese substitutes, and high-protein beverages. While retaining the rich nutritional characteristics of yeast protein, it achieves a comprehensive improvement in bitterness control, texture optimization, and flavor coordination, which is conducive to promoting the innovative development of high-protein microbial dairy products. Detailed Implementation

[0019] The embodiments of the technical solution of the present invention will be described in detail below. These embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore merely examples and should not be used to limit the scope of protection of the present invention.

[0020] Those skilled in the art will understand that the present invention can be practiced even without certain specific details. In some other embodiments, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0021] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, or instruments used, unless otherwise specified by the manufacturer, are all commercially available; and the conditions not specified in the examples are all performed under conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, the raw materials used in this invention are all commercially available products commonly found in this technical field. Unless otherwise specified, the "ratio" referred to in the following examples refers to a ratio of parts by mass.

[0022] It should be specifically stated that the definitions of the amount added and the units of concentration involved in this embodiment are as follows: Material-to-liquid ratio: For example, "1:10 g / mL" means 1 gram of solute (or substrate) is added per 10 mL of solvent, i.e., a weight-to-volume ratio (w / v). Percentage concentration (w / v): For example, "3% (w / v, based on the volume cutoff)" means 3 grams of solute are contained in 100 mL of solution, in g / 100 mL. Enzyme addition percentage: For example, "3% (based on substrate dry weight)" means the percentage of enzyme mass to substrate dry weight (w / w), i.e., 3 grams of enzyme are added per 100 grams of substrate dry weight. Inoculum size: For example, "1×10 8 "CFU / mL" indicates that the number of viable bacteria per milliliter of fermentation substrate is 1 × 10⁻⁶. 8 Colony forming units (CFU). Percentage by weight (w / w): For example, "3.0% high oleic sunflower oil (based on total mass of fermentation substrate)" means that 3.0 grams of additive are added per 100 grams of fermentation substrate, in g / 100g.

[0023] Example 1 The purpose of this embodiment is to provide a method for preparing a yeast protein dairy product, specifically a yeast protein high-protein beverage. The specific preparation method is as follows, including the following steps: S1: Dual-enzyme directed stepwise hydrolysis Weigh 100g of yeast protein raw material (brewer's yeast protein, protein content greater than 60%, sourced from Hubei Guangji Pharmaceutical Co., Ltd., the same batch of raw material was used in other examples and comparative examples), add 1000mL of deionized water, stir evenly to form a mixture system with a material-to-liquid ratio of 1:10 g / mL; adjust the pH of the system to 6.8, stir at 30℃ for 30 minutes to complete the prehydration, and obtain the prehydrated yeast protein mixture; Preliminary enzymatic hydrolysis: Add flavor protease (enzyme activity 50000U / g) to the prehydrated yeast protein mixture at 3% of the dry weight of yeast protein in the prehydrated yeast protein mixture (calculated based on the dry weight of the substrate). Place the reaction system in a 42℃ constant temperature water bath and continuously stir at 100rpm for 90 minutes for hydrolysis. Deep enzymatic hydrolysis: After the initial enzymatic hydrolysis, adjust the pH of the system to 7.1 with 1 mol / L NaOH solution, raise the temperature to 52℃, and add neutral protease (enzyme activity 30000 U / g) to the prehydrated yeast protein mixture at 2% of the dry weight of yeast protein in the prehydrated yeast protein mixture (unless otherwise specified, all calculations are based on the dry weight of the substrate). Continue to stir and hydrolyze at 100 rpm for 30 minutes. After enzymatic hydrolysis, the system was transferred to an 85°C water bath and kept warm for 12 minutes to inactivate the enzyme. Then, it was cooled to 37°C in an ice-water bath to obtain the enzymatic hydrolysate.

[0024] S2: Ultrafiltration Desalination and Concentration A polyethersulfone ultrafiltration membrane (molecular weight cutoff 3 kDa) was selected. The enzymatic hydrolysate was pumped into the ultrafiltration equipment. The operating pressure was controlled at 0.15 MPa, and the solution was circulated and filtered at 30°C until the volume of the solution was concentrated to 50% of the original volume of the enzymatic hydrolysate (approximately 500 mL). The retentate was collected and set aside for later use.

[0025] S3: Compound microbial liquid fermentation Add 2% (w / v, based on the volume of the retentate, in g / 100mL, i.e., 2 grams of lactose per 100 mL of retentate) lactose to the retentate and stir until completely dissolved to obtain the fermentation substrate; autoclave the fermentation substrate at 121°C for 15 minutes and cool to 37°C; then, according to 1×10 8 The compound bacterial agent was inoculated at a ratio of CFU / mL. This compound bacterial agent consisted of L. casei CICC6104, L. delbrueckii subsp. lactis CICC 22185 and P. pentosaceus CICC 22227 with a viable count ratio of 5:2:3. The strains were purchased from the China Industrial Microbial Culture Collection Center (CICC). The liquid at this time is called the base solution.

[0026] The inoculated substrate was placed in a 37°C constant temperature incubator for anaerobic static fermentation for 6 hours, and then fermentation was stopped to obtain the fermented substrate.

[0027] S4: Post-processing and molding Weigh out 3.0% of the total mass of fermentation substrate high oleic sunflower seed oil (oleic acid content ≥70%), preheat at 60℃, add 0.25% sucrose ester S-1670 and 0.15% monoglyceride GMS (both measured by the mass of fermentation substrate), stir until the emulsifier dissolves to form an oil phase system; add the oil phase system to the fermentation substrate, and shear at 20000 rpm for 3 minutes to form an emulsion.

[0028] Add 0.70% high-methoxyl pectin (esterification degree ≥70%) to the emulsion by weight of the emulsion, and stir at 65°C for 10 minutes until dissolved; then dilute the mixture with deionized water to a protein content of 4% (determined by the Kjeldahl method according to GB 5009.5-2016), and then add 0.1% xanthan gum (by weight of the diluted system) and 4% sucrose (by weight of the diluted system), and stir for 10 minutes until completely dispersed.

[0029] Add 0.05% vitamin E (by mass of the diluted system) to the diluted mixture, pasteurize at 85°C for 10 minutes, cool to 25°C and then fill to obtain the yeast protein high-protein beverage.

[0030] It is worth noting that in the aforementioned S4 step, high-oleic sunflower seed oil and high-methoxyl pectin were selected primarily based on their advantages in product texture stability, nutritional characteristics, and process compatibility. High-oleic sunflower seed oil can be replaced with other vegetable oils high in monounsaturated fatty acids, such as low-erucic acid rapeseed oil (oleic acid content 51%~70%, GB 1536-2021), high-oleic peanut oil (oleic acid content ≥75%), or olive oil (oleic acid content 55%~83%). These oils also have good oxidative stability, which can reduce the risk of product oxidative rancidity. High-methoxyl pectin (esterification degree ≥70%) can be replaced with low-methoxyl pectin or natural thickeners such as guar gum and xanthan gum, depending on process requirements. This allows for flexible adjustment of product texture while ensuring the suspension stability of the system.

[0031] Example 2 The purpose of this embodiment is to provide a method for preparing yeast protein dairy products. Unless otherwise specified, the reagents, materials and experimental conditions used in this embodiment and the following comparative examples are consistent with those in Example 1.

[0032] The yeast protein dairy product provided in this embodiment is specifically a yeast protein cheese substitute, and the specific preparation method is as follows, including the following steps: S1: Dual-enzyme directed stepwise hydrolysis Weigh 150g of yeast protein raw material, add 1650mL of deionized water, stir well to form a mixture with a material-to-liquid ratio of 1:11; adjust the pH of the system to 6.5, stir at 30℃ for 25 minutes to complete the prehydration, and obtain the prehydrated yeast protein mixture. Preliminary enzymatic hydrolysis: Add 4% of the dry weight of yeast protein in the prehydrated yeast protein mixture to the flavor protease. Place the reaction system in a 45°C constant temperature water bath and continuously stir at 120 rpm for 120 minutes to hydrolyze. Deep enzymatic hydrolysis: Adjust the pH of the system to 7.2, raise the temperature to 55°C, add neutral protease at 3% of the dry weight of yeast protein in the prehydrated yeast protein mixture, and continue to hydrolyze at 120 rpm for 40 minutes. After enzymatic hydrolysis, the system was transferred to a 90°C water bath and kept warm for 10 minutes to inactivate the enzyme. The solution was then cooled to 37°C to obtain the enzymatic hydrolysate.

[0033] S2: Ultrafiltration Desalination and Concentration A ceramic ultrafiltration membrane (molecular weight cutoff of 3 kDa) was selected. The enzymatic hydrolysate was pumped into the ultrafiltration device. The operating pressure was controlled at 0.2 MPa, and the solution was circulated and filtered at 35°C until the volume of the solution was concentrated to 40% of the original volume (about 600 mL). The filtrate was collected and set aside for later use.

[0034] S3: Compound microbial liquid fermentation Add 3% (w / v, based on the volume of the retentate) lactose to the retentate and stir until completely dissolved to obtain the fermentation substrate. Autoclave the fermentation substrate at 121°C for 15 minutes and cool to 39°C. Inoculate L. casei CICC 6104, L. delbrueckii subsp. lactis CICC 22185, and P. pentosaceus CICC 22227 with a viable cell ratio of 6:3:4, for a total inoculum of 1.2 × 10⁻⁶ cells. 8 CFU / mL; The inoculated substrate was placed in a constant temperature incubator. During fermentation, the initial pH was controlled at 6.0-6.5. Sealed anaerobic culture was used. Under static conditions, the substrate was fermented in a constant temperature incubator at 39℃ for 5 hours. Fermentation was then stopped to obtain the fermented substrate.

[0035] S4: Post-processing and molding Weigh 3.5% of the total mass of the fermentation base with olive oil, preheat it to 65℃, add 0.35% sucrose ester S-1670 and 0.10% succinic acid monoglyceride SMG (based on the mass of the fermentation base), stir until completely dissolved to form an oil phase system; mix the oil phase system with the fermentation base, and emulsify it for 5 minutes at 18000 rpm using a high-speed shear machine to form a homogeneous emulsion.

[0036] Add 0.60% high-methoxyl pectin and 0.30% κ-carrageenan (by mass of emulsion) to the emulsion, and stir in a water bath at 65°C for 15 minutes until the colloids dissolve; adjust the pH of the system to 5.2–5.5 with 1 mol / L NaOH, add rennet at 0.02% (by mass of emulsion), and let stand at 37°C for 30 minutes to allow curd to form.

[0037] Cut the curd into 1cm x 1cm pieces, let it stand at room temperature for 20 minutes to remove the whey, transfer the curd into a mold to shape it, and refrigerate at 4℃ for 24 hours to mature, thus obtaining a yeast protein cheese substitute.

[0038] Example 3 The purpose of this embodiment is to provide a method for preparing a yeast protein dairy product, specifically a stirred yeast protein yogurt. The specific preparation method includes the following steps: S1: Dual-enzyme directed stepwise hydrolysis Weigh 80g of yeast protein raw material, add 720mL of deionized water, stir well to form a mixture with a material-to-liquid ratio of 1:9; adjust the pH of the system to 7.0, stir at 30℃ for 35 minutes to complete the prehydration, and obtain the prehydrated yeast protein mixture. Preliminary enzymatic hydrolysis: Add flavor protease at 2% of the dry weight of yeast protein in the prehydrated yeast protein mixture. Place the reaction system in a 40°C constant temperature water bath and continuously stir at 80 rpm for 60 minutes to hydrolyze. Deep enzymatic hydrolysis: Adjust the pH of the system to 7.0, raise the temperature to 50°C, add neutral protease at 1% of the dry weight of yeast protein in the prehydrated yeast protein mixture, and continue to hydrolyze at 80 rpm for 25 minutes. After enzymatic hydrolysis, the system was transferred to an 80°C water bath and kept warm for 15 minutes to inactivate the enzyme. The solution was then cooled to 37°C to obtain the enzymatic hydrolysate.

[0039] S2: Ultrafiltration Desalination and Concentration An ultrafiltration membrane made of regenerated cellulose (molecular weight cutoff 3 kDa) was selected. The enzymatic hydrolysate was pumped into the ultrafiltration device. The operating pressure was controlled at 0.1 MPa, and the solution was circulated and filtered at 25°C until the volume of the solution was concentrated to 60% of the original volume (approximately 480 mL). The filtrate was collected, and its protein concentration (≥12%) and conductivity (≤550 μS / cm) were tested for later use.

[0040] S3: Compound microbial liquid fermentation Lactose was added to the retentate at a ratio of 1.5% (w / v, g / 100 mL) of the retentate mass and stirred until completely dissolved to obtain the fermentation substrate. The fermentation substrate was autoclaved at 121°C for 15 minutes and cooled to 35°C. L. casei CICC6104, L. delbrueckii subsp. lactis CICC22185, and P. pentosaceus CICC22227 were inoculated at a viable count ratio of 4:1:2, with a total inoculum of 3.1 × 10⁻⁶ cells. 8 CFU / mL; The inoculated substrate was placed in a 35°C constant temperature incubator and allowed to ferment for 8 hours. Fermentation was then stopped to obtain the fermented substrate.

[0041] S4: Post-processing and molding Weigh out 3.0% of the total mass of high-oleic sunflower oil from the fermentation substrate. After preheating at 60℃, add 0.30% sucrose ester S-1670, 0.15% monoglyceride GMS, and 0.05% succinic acid monoglyceride SMG (all based on the mass of the fermentation substrate). Stir until the emulsifier is completely dissolved to form an oil phase system. Slowly add the oil phase system to the fermentation substrate and emulsify it for 3 minutes at 20,000 rpm using a high-speed shear machine to form a uniform emulsion with a particle size of 1–3 μm.

[0042] Add 0.70% high-methoxyl pectin and 0.25% κ-carrageenan (by weight of emulsion) to the emulsion, and stir in a water bath at 65°C for 10 minutes until the colloids are completely dissolved; then add 6% sucrose and 0.08% vitamin E, and stir for 5 minutes until well mixed.

[0043] The mixture is pasteurized at 85°C for 10 minutes, cooled to 25°C, and then poured into aseptic yogurt cups. It is then refrigerated at 4°C for 12 hours to set, thus obtaining stirred yeast protein yogurt.

[0044] Comparative Example 1 The purpose of this comparative example is to observe the effect of the enzymatic hydrolysis process of S1 on the preparation of yeast protein dairy products, specifically: 1.1 Experimental Design and Grouping This comparative example is based on Example 1. By changing the enzymatic digestion method, each group was independently repeated 3 times. The group settings are shown in Table 1: Table 1 Group settings for enzymatic hydrolysis condition testing 1.2 Detection indicators include (1) Bitterness value: The ISO 13299 sensory blind evaluation method was adopted. Ten tasters independently scored the bitterness value from 0 to 10 (0 = no bitterness, 10 = extremely bitterness) and the average value was taken. (2) Sedimentation rate: Centrifuge at 4000 rpm for 15 minutes and calculate the percentage of the precipitate mass to the total protein mass (%). (3) Free amino content: The absorbance was measured at a wavelength of 340 nm using the o-phthalaldehyde method (OPA method) and converted to mg / g protein.

[0045] 1.3 Experimental Results and Analysis The experimental results are shown in Table 2: Table 2. Effects of enzymatic hydrolysis on the preparation of yeast protein dairy products The experimental results above indicate that the stepwise hydrolysis sequence of the two enzymes (flavor enzyme → neutral enzyme) is the core controlling factor for ensuring the hydrolysis efficiency of yeast proteins. Reversing the hydrolysis sequence or repeated hydrolysis with a single enzyme significantly exacerbates the formation of bitter peptides and reduces protein solubility. While simultaneous addition of two enzymes can increase the depth of hydrolysis, it cannot achieve synergistic optimization of bitterness control and system stability. The speculated mechanism is that the flavor enzyme preferentially acts on the hydrophilic peptide bonds on the protein surface, initially breaking them and exposing the internal hydrophobic region, creating conditions for deep hydrolysis by the neutral enzyme. If the sequence is reversed, the neutral enzyme directly attacks the hydrophobic groups, leading to the formation of a large number of bitter peptides, such as proline residue-enriched fragments, while competition for hydrolysis sites reduces hydrolysis efficiency. Experimental data show that although the simultaneous addition group had the highest free amino content, the precipitation rate was 128% higher than the control group, confirming that stepwise hydrolysis achieves a synergistic effect of "removing bitter precursors first → breaking the hydrophobic core" through temporal separation. In addition, the precipitation rate (14.5%) of groups 1-5 (secondary flavor enzymatic hydrolysis) was significantly higher than that of the control group, and the free amino content decreased by 20.5%, indicating that flavor protease cannot replace the ability of neutral enzyme to deeply hydrolyze hydrophobic regions, verifying the necessity of the stepwise enzymatic hydrolysis sequence of the two enzymes.

[0046] Comparative Example 2 The purpose of this comparative example is to observe the effect of the S2 retention threshold on the preparation of yeast protein dairy products, specifically: 2.1 Experimental Design and Grouping This comparative study investigated the effect of adjusting the molecular weight cutoff of the ultrafiltration membrane on desalination, debittering, and system stability. The group settings are shown in Table 3. Table 3 Group settings for ultrafiltration retention 2.2 Detection indicators include (1) Na⁺ residue: Sodium ions are the main contributor to the saltiness of dairy products, and their content directly affects the palatability of the product. Therefore, ion chromatography (GB 5009.91) was used to detect the sodium ion concentration (mg / kg) in the prepared dairy products. (2) Bitter peptide content: Due to the imine ring structure of the side chain, proline can enhance the spatial rigidity of the peptide chain, resulting in a significant increase in bitterness. Therefore, based on the good retention ability of the C18 column for hydrophobic compounds, the HPLC method (C18 column, 220nm UV detection) was used to quantify the content of proline oligopeptides (μg / g, based on sample mass) in dairy products to characterize the bitter peptide content. (3) Particle size distribution (PDI): Particle size distribution directly relates to the risk of aggregation of the system by characterizing the uniformity of particle size distribution. The smaller the PDI value (usually <0.3 is the ideal state), the higher the uniformity of particle size. The smaller the difference in sedimentation rate between particles due to size differences, the more effectively gravity stratification or flocculation can be avoided. Therefore, the uniformity of the system is characterized by measuring the particle size distribution index of the system by dynamic light scattering (DLS). The lower the PDI value, the more stable the system is generally.

[0047] 2.3 Experimental Results and Analysis The experimental results are shown in Table 4: Table 4. Effect of ultrafiltration cutoff threshold on the preparation of yeast protein dairy products The choice of molecular weight cutoff for ultrafiltration directly affects the flavor and textural stability of the product. The experimental results above indicate that 3kDa, as the optimal threshold, balances desalination efficiency and bitter peptide removal, achieving a desalination-debitterness balance. A molecular weight cutoff that is too small leads to increased loss of target proteins and a lower PDI (particulate ionization index) compared to the 3kDa group. Conversely, a molecular weight cutoff that is too large fails to effectively remove small-molecule salty ions and bitter peptides, resulting in system aggregation. The non-ultrafiltration group exhibited poor flavor and stability, with unacceptable Na+ residue levels.

[0048] Comparative Example 3 The purpose of this comparative study is to observe and verify the effects of the ratio and function of the compound strains on the preparation of yeast protein dairy products. Specifically: 3.1 Experimental Design and Grouping This comparative example, by setting up single-strain groups, imbalanced ratio groups, and missing-strain groups, verified whether there was a synergistic effect among the compound inoculum (L. casei :L. lactis :P. pentosaceus=5:2:3). The amount of fermentation bacteria used in this comparative example was kept consistent (total inoculum 1×10⁻⁶). 8 (CFU / mL), the bacterial strain combinations and their ratios were varied, and the group settings are shown in Table 5: Table 5. Proportion of Compound Microbial Strains and Grouping for Microbial Function Verification 3.2 Detection indicators include (1) Endpoint pH: The acidity of the system at the end of fermentation was monitored in real time using a pH meter; (2) Acetaldehyde content: Acetaldehyde is the core source of the characteristic flavor of fermented milk. Therefore, the concentration of acetaldehyde (mg / kg) of characteristic flavor substances was determined by headspace-GC / MS. (3) Odor rating: A blind sensory evaluation panel of 10 people evaluated the intensity of yeast fishy / bitter taste on a scale of 0-10 (0 = no odor, 10 = extremely strong odor).

[0049] 3.3 Experimental Results and Analysis The experimental results are shown in Table 6: Table 6. Effects of the ratio and function of the compound strains on the preparation of yeast protein dairy products. The above experimental results indicate that fermentation with a specific ratio of viable microorganisms exhibits a significant synergistic effect. Specifically, *L. casei* dominates the acid production process, maintaining the pH of the system stable between 4.0 and 4.6 through lactic acid generation; *L. lactis* utilizes pyruvate decarboxylase to specifically synthesize acetaldehyde (the control group reached 15.3 mg / kg, 1.2 times the highest value in the single-strain group); *P. pentosaceus* breaks down fats through esterase, inhibiting the accumulation of yeast odor substances (such as hexanal). In the single-strain group, fermentation with *L. casei* alone may lead to excessively low pH, potentially causing protein denaturation, while fermentation with *P. pentosaceus* alone results in insufficient acid production, leading to an off-odor score of 4.2. After adjusting the ratio, the acetaldehyde content in groups 3-4 decreased by 38%, confirming that an imbalance in the microbial ratio disrupts the synergistic effect of metabolic pathways. Only a specific ratio combination can simultaneously achieve the triple goals of acidity control, flavor synthesis, and off-odor suppression.

[0050] Comparative Example 4 The purpose of this comparative example is to observe the effect of the emulsifier compound system on the preparation of yeast protein dairy products, specifically: 4.1 Experimental Design and Grouping This comparative example used a fixed oil phase (3% high-oleic sunflower oil) and shear conditions (20,000 rpm × 3 minutes) to verify the stabilizing effect of the compound system (0.25% sucrose ester + 0.15% monoglyceride) on the prepared liquid dairy products by adjusting the composition and ratio of emulsifiers. The experimental groups are shown in Table 7. Table 7. Experimental Grouping of Emulsifier Compound System 4.2 Detection indicators include Emulsifying Activity Index (EAI, m) 2 / g): Initial emulsifying capacity was calculated by turbidimetric method (absorbance at 500 nm); Emulsion stability (ESI, %): Anti-stratification ability was evaluated by centrifugation (4000 rpm × 30 min) combined with image analysis; Interfacial protein adsorption capacity (mg / m³) 2 The amount of emulsifier-protein adsorbed at the interface was determined using a dissipative quartz microcrystalline balance (QCM-D).

[0051] 4.3 Experimental Results and Analysis The experimental results are shown in Table 8: Table 8. Effects of emulsifier compound system composition on the preparation of yeast protein dairy products. The above experimental results indicate that the composition ratio of the emulsifier compound system is the core factor in ensuring emulsion stability. Using a single emulsifier or reversing the ratio leads to a decrease in initial emulsifying ability and weakened anti-stratification performance. Meanwhile, the ESI (94.5%) of the control group (0.25% sucrose ester + 0.15% monoglyceride) was 9.4% higher than that of group 4-4 (equal proportion compound), and the interfacial protein adsorption was increased by 23.4%, indicating that a slightly higher sucrose ester ratio makes it easier to form a stable interfacial film with proteins. The compound system of 0.25% sucrose ester and 0.15% monoglyceride can synergistically increase the adsorption of interfacial proteins, forming a stable emulsion interfacial film, effectively inhibiting oil phase aggregation, and thus ensuring the long-term physical stability of the product.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a yeast protein dairy product, characterized in that, Includes the following steps: S1: Dual-enzyme directed stepwise hydrolysis After prehydration, the yeast protein raw material is first subjected to preliminary enzymatic hydrolysis using flavor protease, and then further enzymatic hydrolysis using neutral protease. After the enzymatic hydrolysis is completed, the enzyme is inactivated and cooled to obtain the hydrolysate. S2: Ultrafiltration Desalination and Concentration The enzymatic hydrolysate from step S1 was filtered using an ultrafiltration membrane to retain target proteins with a molecular weight >3 kDa, while removing small peptides and salt ions to obtain the retained product. S3: Compound microbial liquid fermentation Lactose was added to the retentate from step S2 as a fermentation substrate. After sterilization, a compound microbial agent composed of lactic acid bacteria and Pediococcus was inoculated and allowed to ferment statically. The pH at the end of the fermentation was controlled to be 4.0–4.6 to obtain the fermentation substrate. S4: Post-processing and product molding The fermentation substrate is subjected to molding treatment to obtain yeast protein dairy products.

2. The method for preparing a yeast protein dairy product as described in claim 1, characterized in that: In S1, the prehydration process of yeast protein raw material involves adjusting the pH of the system to 6.5–7.0 using a pH adjuster, and the ratio of yeast protein raw material to solvent is 1:9–11.

3. The method for preparing a yeast protein dairy product as described in claim 1, characterized in that: The enzymatic hydrolysis conditions for flavor proteases are as follows: Preliminary enzymatic hydrolysis: Enzymatic hydrolysis was performed using flavor protease. The hydrolysis conditions were as follows: flavor protease was added at a mass ratio of 2%-4% to substrate, and the reaction system was placed in a constant temperature water bath at 40-45℃ and continuously stirred at a speed of 80-120 rpm for 50-150 minutes. Deep enzymatic hydrolysis: After the initial enzymatic hydrolysis is completed, adjust the pH of the system to 7.0-7.2, raise the temperature to 50-55℃, add neutral protease at an enzyme to substrate mass ratio of 1%-3%, and continue stirring and hydrolyzing for 20-50 minutes; After the reaction is complete, enzyme inactivation treatment is performed.

4. The method for preparing a yeast protein dairy product as described in claim 1, characterized in that: The ultrafiltration membrane in S2 is made of polyethersulfone, regenerated cellulose, or ceramic, and operates at a pressure of 0.1–0.2 MPa.

5. The method for preparing a yeast protein dairy product as described in claim 1, characterized in that: The compound microbial agent in S3 is composed of lactic acid bacteria and Pediococcus pentosaceus, wherein the lactic acid bacteria are selected from at least one of Lactobacillus casei and Lactobacillus delbrueckii.

6. The method for preparing a yeast protein dairy product as described in claim 1, characterized in that: The compound microbial agent is composed of L. casei CICC 6104, L. delbrueckii subsp. lactis CICC 22185 and P. pentosaceus CICC 22227 in a live bacteria ratio of 4-6:1-3:2-4. The fermentation temperature is 35–39℃, and the fermentation time is 5–8 hours.

7. The method for preparing a yeast protein dairy product as described in claim 1, characterized in that, S4 specifically involves: mixing the fermentation base material with the system containing the oil phase and emulsifier, performing high-speed shear emulsification, adding a colloidal stabilizer, sterilizing, and then molding according to the product type.

8. The method for preparing a yeast protein dairy product as described in claim 7, characterized in that: The oil phase is a vegetable oil, selected from at least one of sunflower seed oil, olive oil, and soybean oil; The emulsifier comprises at least one of sucrose ester, monoglyceride, and monoglyceride succinate; The colloidal stabilizer is a compound of at least two of pectin, carrageenan, and xanthan gum.

9. A yeast protein dairy product prepared by the method according to any one of claims 1–8.

10. The yeast protein dairy product as described in claim 9, characterized in that: The yeast protein dairy product is any one of stirred yogurt, cheese substitute, or high-protein beverage.

Images