A method for preparing high-protein yeast extract by synergistically breaking cell walls and stepwise enzymolysis

Abstract

The application provides a high-protein yeast extract preparation method combining cell wall breaking and step-by-step enzymolysis, and belongs to the technical field of bioengineering and food ingredients. The yeast extract preparation method comprises the following steps: S1. centrifuging yeast fermentation liquor to obtain wet slurry; S2. mixing the wet slurry with water to form a suspension, and homogenously treating the suspension to obtain slurry; S3. adding a compound cell wall breaking enzyme to the slurry for enzymolysis, and then adding a neutral protease for enzymolysis; S4. inactivating after the enzymolysis is completed, and adding water for extraction; S5. performing solid-liquid separation on the extracted material, and collecting supernatant; and S6. concentrating the obtained supernatant to obtain yeast extract. The preparation method can effectively increase the protein content in the yeast extract. Moreover, the preparation method has the advantages of short production cycle, mild and controllable conditions, reasonable process, suitability for industrialization, and good industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and food ingredient technology, specifically relating to a method for preparing high-protein yeast extract through synergistic cell wall disruption and stepwise enzymatic hydrolysis. Background Technology

[0002] Yeast extract is a functional raw material made from yeast cells through steps such as cell wall disruption, autolysis or exogenous enzymatic hydrolysis, solid-liquid separation, and concentration. It is widely used in food flavoring, fermentation media, and bio-fermentation aids. With the increasing demand for high-protein, high-utilization-rate raw materials in the food industry and bio-fermentation industry, high-protein yeast extract is gradually becoming an important development direction.

[0003] Existing yeast extract preparation technologies mainly include autolysis, single or simple combinations of enzymatic hydrolysis, and methods combining mechanical disruption and enzymatic hydrolysis. However, existing technologies generally suffer from the following problems: First, process evaluation indicators fail to accurately reflect protein utilization efficiency. Existing processes often use "yeast extract yield" or "dry-based protein content of extract" as primary evaluation indicators. However, these indicators only reflect the apparent composition of the product and cannot truly reflect the conversion and utilization efficiency of the raw material protein during the preparation process. High yield or high dry-based protein content does not equate to the raw material protein being fully converted and entering the target product system, potentially causing process optimization to deviate from the essential goal of efficient protein utilization.

[0004] Second, the cell wall disruption and enzymatic hydrolysis steps lack synergistic design. Current technologies often simply superimpose these steps, lacking a synergistic design tailored to the structural characteristics of yeast cell walls. Yeast cell walls have a complex, multi-layered structure; insufficient disruption will hinder the release of intracellular proteins. Furthermore, if the enzymatic hydrolysis conditions do not match the disruption state, the released proteins may not effectively enter the soluble phase, resulting in a waste of protein resources.

[0005] Third, there is a lack of quantitative definition of protein conversion efficiency. Existing publicly available technologies rarely provide a clear definition and quantitative description of the "proportion of raw material protein entering the yeast extract system," making it difficult to objectively compare different process routes in terms of protein utilization efficiency. This hinders the improvement of industry technology and the scientific selection of process routes.

[0006] Fourth, the process is not adaptable enough to fluctuations in raw material content. Under industrial scale-up conditions, the protein content of yeast raw materials fluctuates due to factors such as fermentation batches and strain differences. Some existing processes are poorly adapted to this, resulting in insufficient product quality stability and difficulty in meeting the quality consistency requirements of continuous industrial production.

[0007] In summary, there is an urgent need for a process method that can increase the proportion of raw material protein entering the yeast extract system and stably prepare high-protein yeast extract under conditions where the raw material protein content fluctuates to a certain extent. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing high-protein yeast extract through synergistic cell disruption and stepwise enzymatic hydrolysis. Using yeast cells as raw material, the method promotes the full release and dissolution of intracellular yeast proteins through the synergistic effect of physical cell disruption and stepwise enzymatic hydrolysis, allowing as much of the raw material protein as possible to enter the yeast extract system. This ensures the protein content of the product while improving the overall utilization efficiency of the raw material protein.

[0009] This invention provides a method for preparing yeast extract, comprising the following steps: S1. Centrifuge the yeast fermentation broth to obtain wet bacterial sludge; S2. Mix the wet bacterial mud with water to form a suspension, and then homogenize and break down the cell walls of the suspension to obtain a slurry; S3. Add compound cell wall-breaking enzyme to the slurry for enzymatic hydrolysis, and then add neutral protease for enzymatic hydrolysis; S4. After enzymatic hydrolysis, the enzyme is inactivated and then water is added for extraction; S5. Perform solid-liquid separation on the extracted material and collect the supernatant; S6. Concentrate the obtained supernatant to obtain yeast extract.

[0010] Furthermore, In step S1, the wet bacterial sludge is washed with water after centrifugation and then centrifuged again. And / or, in step S2, the mass ratio of the wet bacterial mud to water is (6-8):(4-2); And / or, in step S2, the pressure of the homogenization is 60-120 MPa.

[0011] Furthermore, In step S1, the centrifugation speed is 6000-8000 rpm, and the centrifugation time is 10-30 minutes; And / or, in step S2, the homogenization pressure is 80-100 MPa, and the homogenization is performed 1-3 times.

[0012] Furthermore, In step S3, before adding the compound cell wall-breaking enzyme, the temperature of the slurry is adjusted to 55-65℃ and the pH is adjusted to 5.0-6.0; And / or, in step S3, the composite cell-wall-breaking enzyme is composed of cell-wall-breaking enzyme, dextranase, and mannanase in a mass ratio of (1-3):(0.5-1):(0.5-1); And / or, in step S3, the amount of the compound cell-wall-breaking enzyme added is 0.2-0.4% of the dry weight of yeast; And / or, in step S3, the addition of the compound cell-wall-breaking enzyme for enzymatic hydrolysis is carried out by stirring at 60-65℃ and 100-500rpm for 3-4 hours.

[0013] Furthermore, in step S3, the mass ratio of the cell wall-breaking enzyme, dextranase, and mannanase is 2:0.8:0.8.

[0014] Furthermore, In step S3, the neutral protease is Bacillus subtilis neutral protease; And / or, in step S3, the amount of neutral protease added is 0.4-0.6% of the dry weight of yeast; And / or, in step S3, the addition of neutral protease for enzymatic hydrolysis is performed by stirring at pH 5.0-6.0, 60-65℃, and 100-500 rpm for 4-6 hours.

[0015] Furthermore, In step S4, the inactivation is performed by incubating at 80-95°C for 10-30 minutes to inactivate the enzyme; And / or, in step S4, the volume of the water is 1-2 times the volume of the inactivated enzymatic hydrolysate; And / or, in step S4, the extraction is performed by stirring at 35-45°C for 0.5-1 hour; And / or, in step S6, the concentration is carried out under vacuum conditions of -0.080 to -0.090 MPa and 55-65°C until the solid content reaches 55%-65%.

[0016] Further, in step S4, before inactivation, the system is adjusted to a temperature of 45-55℃ and a pH of 6.5-7.5, and flavor protease is added for treatment for 1-2 hours. The amount of flavor protease added is 0.05%-0.2% of the dry weight of yeast.

[0017] The present invention also provides a yeast extract, which is prepared by the aforementioned yeast extract preparation method.

[0018] The present invention also provides the use of the aforementioned yeast extract in the fields of food flavoring, fermentation culture medium, and biological fermentation aid.

[0019] This invention aims to address the problems of low overall protein conversion efficiency and insufficient stability of product protein content in existing yeast extract preparation processes. By providing a preparation method with strong process synergy, the proportion of protein in the raw yeast entering the final extract product (i.e., protein extraction rate) is significantly increased, thereby stably preparing high-protein yeast extract even under conditions where the protein content of the raw material fluctuates.

[0020] The technical features of this invention are as follows: Innovation in process objectives: The "protein extraction rate" is clearly defined as the core process evaluation indicator. This indicator is defined as (quality of yeast extract × dry basis protein content of extract) ÷ (quality of raw yeast × dry basis protein content of raw material) × 100%, which directly reflects the conversion and utilization efficiency of raw material protein.

[0021] Collaborative design of process steps: "Collaborative cell disruption": Using specific high-pressure homogenization conditions to physically disrupt cells, creating sufficient contact conditions for subsequent enzymatic hydrolysis.

[0022] "Stepwise enzymatic hydrolysis": This innovative approach divides the enzymatic hydrolysis process into two stages: the first stage focuses on the degradation of residual cell wall structures, while the second stage concentrates on the hydrolysis of released proteins. This stepwise strategy avoids interference or substrate competition between different enzyme systems, significantly improving overall efficiency.

[0023] "Inactivation followed by water extraction": After enzyme inactivation and before solid-liquid separation, a gentle water extraction step is introduced. This step effectively promotes the diffusion and distribution of products that have been enzymatically hydrolyzed into soluble peptides and amino acids from the solid phase of cell debris to the aqueous phase, and is one of the key steps to improve protein extraction rate.

[0024] Compared with the prior art, the advantages and positive effects of the present invention are as follows: (1) High utilization rate of raw material protein: Through the synergistic effect of the above three steps, the "protein extraction rate" can be significantly improved. In the common range of raw material dry basis protein content of 45%-65%, the protein extraction rate can reach 50%-70%, which is much higher than the traditional autolysis method (usually <35%) and simple mixed enzymatic hydrolysis method (usually <45%).

[0025] (2) High and stable cell wall breakage rate: The cell wall breakage rate is consistently above 98%, as verified by microscopic counting method.

[0026] (3) Short production cycle: The core enzymatic hydrolysis time is only 7-10 hours, which is a significant improvement compared to traditional enzymatic hydrolysis (>24 hours).

[0027] (4) Mild and controllable conditions: pH 5.0-6.0 and temperature 60-65℃ throughout the process to avoid protein denaturation.

[0028] (5) High and stable protein content: It can stably prepare high-protein yeast extract with a dry basis protein content of 55%-70%, and the product quality is superior and more adaptable to raw material fluctuations.

[0029] (6) The process is reasonable and suitable for industrialization: the conditions of each step are clear, the controllability is strong, and it is easy to modify and implement on the existing yeast extract production line, which has good industrialization prospects.

[0030] In summary, this invention provides a method for preparing high-protein yeast extract, which can effectively increase the protein content in yeast extract. Furthermore, this method features a short production cycle and mild, controllable conditions. The process is rational and suitable for industrialization, possessing promising prospects for commercialization.

[0031] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0032] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the method for preparing high-protein yeast extract according to the present invention. Detailed Implementation

[0034] Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0035] The yeast fermentation broth used in this embodiment of the invention was obtained using a conventional multi-stage expansion method in the art: the yeast strain was cultured in shake flasks and then progressively scaled up in a seed tank before being transferred to a fermenter for fermentation, during which carbon and nitrogen sources were added; once the cell concentration reached the target, the yeast fermentation broth was harvested. Furthermore, the yeast fermentation broth in this invention can also be obtained or purchased using other methods in the art.

[0036] A schematic diagram of the method for preparing high-protein yeast extract of the present invention is shown below. Figure 1 As shown. The process of this invention is as follows: S1. Raw material collection and washing: Centrifuge the yeast fermentation broth at 6000-8000 rpm for 10-30 minutes and collect the resulting wet bacterial sludge; wash the wet bacterial sludge once with deionized water to remove some fermentation residue.

[0037] S2. Suspension preparation and physical cell wall disruption: Mix wet bacterial sludge and purified water at a mass ratio of (6-8):(4-2) to form a suspension. Perform high-pressure homogenization and cell wall disruption treatment on the suspension. The homogenization pressure is 60-120 MPa, preferably 80-100 MPa, and the mixture is circulated 1-3 times, preferably 2 times.

[0038] S3. Stepwise complex enzymatic hydrolysis: Adjust the temperature of the homogenized slurry to 55-65℃.

[0039] S3a. First stage of enzymatic hydrolysis: Add the compound cell-wall-breaking enzyme to the yeast homogenate and hydrolyze for 3-4 hours at 200 rpm under conditions of pH 5.0-6.0 and temperature 60-65℃. The compound cell-wall-breaking enzyme is composed of cell-wall-breaking enzyme, glucanase, and mannanase in a mass ratio of (1-3):(0.5-1):(0.5-1). The amount of compound cell-wall-breaking enzyme added (by weight) is 0.2-0.4% of the dry weight of yeast.

[0040] S3b. Second stage of enzymatic hydrolysis: Add neutral protease directly without changing the system conditions, and continue enzymatic hydrolysis at pH 5.0-6.0 and temperature 60-65℃ with stirring at 200 rpm for 4-6 hours. The amount of neutral protease added (by weight) is 0.4%~0.6% of the dry weight of yeast.

[0041] S4. Inactivation and Water Extraction: After enzymatic hydrolysis, heat to 80-95℃ and incubate for 10-30 minutes to inactivate the enzyme. Then, add 1-2 times the mass of purified water to the inactivated enzyme hydrolysate and stir at 35-45℃ for 0.5-1 hour.

[0042] S5. Solid-liquid separation: The material after water extraction is subjected to solid-liquid separation (e.g., centrifugation), and the supernatant is collected. Preferably, the separated solid phase is washed 1-2 times with purified water, and the washing liquid and supernatant are combined.

[0043] S6. Concentration: Combine all liquid phases and concentrate them under vacuum conditions of -0.080 to -0.090 MPa and 55-65℃ until the solid content reaches 55%-65%, thus obtaining the high-protein yeast extract product.

[0044] After step S3 is completed and before enzyme inactivation, the system can be adjusted to a temperature of 45-55℃ and a pH of 6.5-7.5, and flavor protease can be added for 1-2 hours. The amount of flavor protease added (by weight) is 0.05%-0.2% of the dry weight of yeast.

[0045] The above-mentioned dry weight of yeast refers to the weight of the substance obtained after centrifugation and sedimentation of yeast fermentation broth, and drying of the resulting wet mycelium sludge to constant weight.

[0046] The following embodiments and comparative examples of the present invention use the same batch of yeast fermentation broth.

[0047] Example 1: Preparation of a high-protein yeast extract S1. Raw Material Preparation A batch of yeast fermentation broth was collected, centrifuged (6000 rpm for 30 minutes), washed with purified water, and the wet mycelium sludge was collected. The dry-basis protein content of this batch of yeast raw material was determined to be 52%. A wet mycelium sludge equivalent to 100.0 kg of dry basis (approximately 80% moisture content, total weight approximately 500 kg) was used for subsequent experiments. The total protein content of this batch of raw material was 100 kg × 52% = 52.0 kg.

[0048] S2. Suspension preparation and cell wall disruption treatment The above-mentioned wet bacterial sludge was mixed with purified water at a mass ratio of 7:3 and stirred to prepare a uniform suspension. The suspension was then subjected to cell wall disruption treatment using a high-pressure homogenizer at a pressure of 90 MPa, and the process was repeated twice to obtain a yeast homogenate.

[0049] S3. Stepwise complex enzymatic hydrolysis Adjust the temperature of the yeast homogenate to 60℃ (water bath), and adjust the pH to 5.5 using HCl or NaOH aqueous solution.

[0050] First stage of enzymatic hydrolysis: Add a compound cell wall disrupting enzyme (mass ratio of cell wall disrupting enzyme: β-glucanase: mannanase = 2:0.8:0.8) to the yeast homogenate. The amount of compound cell wall disrupting enzyme added (by weight) is 0.25% of the dry weight of the yeast. Enzymatic hydrolysis is carried out at 60℃ and 200 rpm for 3.5 hours.

[0051] In this invention, the cell wall-breaking enzyme, β-glucanase, and mannanase were all purchased from Beijing Keweibo Biotechnology Co., Ltd. The production batch number of the cell wall-breaking enzyme was YM1250719.

[0052] The second stage of enzymatic hydrolysis involved directly adding Bacillus subtilis neutral protease at a concentration of 0.45% of the yeast dry weight. Hydrolysis was continued for 5 hours under the same conditions (60°C, 200 rpm). The Bacillus subtilis neutral protease was purchased from Longkote Enzyme Preparations.

[0053] S4. Inactivation and water extraction After enzymatic hydrolysis, heat to 92℃ and incubate for 15 minutes to inactivate the enzyme. Add an equal mass of purified water and stir at 40℃ for 40 minutes.

[0054] S5. Solid-liquid separation and washing Centrifuge (6000 rpm, 30 min) and collect the supernatant. Wash the solid phase once with purified water and combine the wash solution with the supernatant.

[0055] S6. Concentrated The high-protein yeast extract was concentrated to a solid content of 60% under a vacuum of -0.085 MPa and a temperature of 62°C to obtain the extract.

[0056] Effect evaluation: 91.7 kg of yeast extract product was obtained (60% solids content, 55.0 kg on dry basis).

[0057] Dry basis yield = 55.0 kg / 100.0 kg × 100% = 55.0%.

[0058] The product has a dry-basis protein content of 58.0%.

[0059] The protein content entering the product = 55.0 kg × 58.0% = 31.9 kg.

[0060] Protein extraction rate = 31.9 kg / 52.0 kg × 100% = 61.3%.

[0061] Example 2: Low Enzyme Addition Process S1. Raw Material Preparation Same as Example 1.

[0062] S2. Suspension preparation and cell wall disruption treatment The above-mentioned wet bacterial sludge was mixed with purified water at a mass ratio of 7:3 and stirred to prepare a uniform suspension. The suspension was then subjected to cell wall disruption treatment using a high-pressure homogenizer at a pressure of 90 MPa, and the process was repeated twice to obtain a yeast homogenate.

[0063] S3. Stepwise complex enzymatic hydrolysis Adjust the temperature of the yeast homogenate to 60℃ (water bath), and adjust the pH to 5.5 using HCl or NaOH aqueous solution.

[0064] First stage of enzymatic hydrolysis: A compound cell-wall-breaking enzyme (mass ratio of cell-wall-breaking enzyme: β-glucanase: mannanase = 2:0.8:0.8) was added to the yeast homogenate. The amount of compound cell-wall-breaking enzyme added was 0.2% of the dry weight of the yeast. Enzymatic hydrolysis was carried out at 60°C and 200 rpm for 4 hours. The sources of the cell-wall-breaking enzyme, β-glucanase, and mannanase were the same as in Example 1.

[0065] Second stage of enzymatic hydrolysis: Bacillus subtilis neutral protease was added directly. The amount of Bacillus subtilis neutral protease added (by weight) was 0.4% of the dry weight of yeast. Enzymatic hydrolysis was continued for 6 hours under the same conditions (60°C, 200 rpm). The source of Bacillus subtilis neutral protease was the same as in Example 1.

[0066] S4. Inactivation and water extraction Same as Example 1.

[0067] S5. Solid-liquid separation and washing Same as Example 1.

[0068] S6. Concentrated Same as Example 1.

[0069] Effect evaluation: 88.7 kg of yeast extract product was obtained (60% solids content, 53.2 kg on dry basis).

[0070] Dry basis yield = 53.2 kg / 100.0 kg × 100% = 53.2%.

[0071] The product has a dry-basis protein content of 56.5%.

[0072] The protein content entering the product = 53.2 kg × 56.5% = 30.06 kg.

[0073] Protein extraction rate = 30.06 kg / 52.0 kg × 100% = 57.8%.

[0074] Example 3: High Enzyme Addition Rate Process S1. Raw Material Preparation Same as Example 1.

[0075] S2. Suspension preparation and cell wall disruption treatment The above-mentioned wet bacterial sludge was mixed with purified water at a mass ratio of 7:3 and stirred to prepare a uniform suspension. The suspension was then subjected to cell wall disruption treatment using a high-pressure homogenizer at a pressure of 90 MPa, and the process was repeated twice to obtain a yeast homogenate.

[0076] S3. Stepwise complex enzymatic hydrolysis Adjust the temperature of the yeast homogenate to 65℃ (water bath), and adjust the pH to 5.5 using HCl or NaOH aqueous solution.

[0077] First stage of enzymatic hydrolysis: A compound cell-wall-breaking enzyme (mass ratio of cell-wall-breaking enzyme: β-glucanase: mannanase = 2:0.8:0.8) was added to the yeast homogenate. The amount of compound cell-wall-breaking enzyme added was 0.25% of the dry weight of the yeast. Enzymatic hydrolysis was carried out at 60°C and 200 rpm for 3 hours. The sources of the cell-wall-breaking enzyme, β-glucanase, and mannanase were the same as in Example 1.

[0078] Second stage of enzymatic hydrolysis: Bacillus subtilis neutral protease was added directly. The amount of Bacillus subtilis neutral protease added (by weight) was 0.6% of the dry weight of yeast. Enzymatic hydrolysis was continued for 4 hours under the same conditions (60°C, 200 rpm). The source of Bacillus subtilis neutral protease was the same as in Example 1.

[0079] S4. Inactivation and water extraction Same as Example 1.

[0080] S5. Solid-liquid separation and washing Same as Example 1.

[0081] S6. Concentrated Same as Example 1.

[0082] Effect evaluation: 94.7 kg of yeast extract product was obtained (60% solids content, 56.8 kg on dry basis).

[0083] Dry basis yield = 56.8 kg / 100.0 kg × 100% = 56.8%.

[0084] The product has a dry-basis protein content of 59.5%.

[0085] The protein content entering the product = 56.8 kg × 59.5% = 33.80 kg.

[0086] Protein extraction rate = 33.80 kg / 52.0 kg × 100% = 65.0%.

[0087] Example 4: Process of adding flavor protease S1. Raw Material Preparation Same as Example 1.

[0088] S2. Suspension preparation and cell wall disruption treatment Same as Example 1.

[0089] S3. Stepwise complex enzymatic hydrolysis Adjust the temperature of the yeast homogenate to 60℃ (water bath) and set the pH to 5.5.

[0090] First enzymatic hydrolysis: Same as in Example 1 (the amount (by weight) of the compound cell wall-breaking enzyme added is 0.25% of the dry weight of yeast, for 3.5 hours).

[0091] Second enzymatic hydrolysis: Same as in Example 1 (the amount (by weight) of Bacillus subtilis neutral protease added is 0.45% of the dry weight of yeast, 5 hours).

[0092] After the second stage of enzymatic hydrolysis, the system was adjusted to a temperature of 50°C and a pH of 7.0. Flavor protease was then added (at a rate of 0.1% of the dry weight of the yeast), and enzymatic hydrolysis continued for 1.5 hours. The flavor protease was purchased from Longkote Enzyme Preparations.

[0093] S4. Inactivation and water extraction After enzymatic hydrolysis, heat to 92℃ and incubate for 15 minutes to inactivate the enzyme. Add an equal mass of purified water and stir at 40℃ for 40 minutes.

[0094] S5. Solid-liquid separation and washing Same as Example 1.

[0095] S6. Concentrated Same as Example 1.

[0096] Effect evaluation: 92.5 kg of yeast extract product was obtained (60% solids content, 55.5 kg on dry basis).

[0097] Dry basis yield = 55.5 kg / 100.0 kg × 100% = 55.5%.

[0098] The product has a dry-basis protein content of 58.5%.

[0099] The protein content entering the product = 55.5 kg × 58.5% = 32.47 kg.

[0100] Protein extraction rate = 32.47 kg / 52.0 kg × 100% = 62.4%.

[0101] Example 5: High-temperature short-time enzymatic hydrolysis process S1. Raw Material Preparation Same as Example 1.

[0102] S2. Suspension preparation and cell wall disruption treatment Same as Example 1.

[0103] S3. Stepwise complex enzymatic hydrolysis Adjust the temperature of the yeast homogenate to 65℃ (water bath), and adjust the pH to 5.5 using HCl or NaOH aqueous solution.

[0104] First stage of enzymatic hydrolysis: A compound cell-wall-breaking enzyme (mass ratio of cell-wall-breaking enzyme: β-glucanase: mannanase = 2:0.8:0.8) was added to the yeast homogenate. The amount of compound cell-wall-breaking enzyme added was 0.4% of the dry weight of the yeast. Enzymatic hydrolysis was carried out at 60°C and 200 rpm for 3 hours. The sources of the cell-wall-breaking enzyme, β-glucanase, and mannanase were the same as in Example 1.

[0105] Second stage of enzymatic hydrolysis: Bacillus subtilis neutral protease was added directly. The amount of Bacillus subtilis neutral protease added (by weight) was 0.6% of the dry weight of yeast. Enzymatic hydrolysis was continued for 4 hours under the same conditions (60°C, 200 rpm). The source of Bacillus subtilis neutral protease was the same as in Example 1.

[0106] S4. Inactivation and water extraction Same as Example 1.

[0107] S5. Solid-liquid separation and washing Same as Example 1.

[0108] S6. Concentrated Same as Example 1.

[0109] Effect evaluation: 90.8 kg of yeast extract product was obtained (60% solids content, 54.5 kg on dry basis).

[0110] Dry basis yield = 54.5 kg / 100.0 kg × 100% = 54.5%.

[0111] The product has a dry-basis protein content of 57.2%.

[0112] The protein content entering the product = 54.5 kg × 57.2% = 31.17 kg.

[0113] Protein extraction rate = 31.17 kg / 52.0 kg × 100% = 59.9%.

[0114] Example 6: Low Homogeneous Pressure Process S1. Raw Material Preparation Same as Example 1.

[0115] S2. Suspension preparation and cell wall disruption treatment: Same as Example 1, except the homogenization pressure is 60 MPa, and the cycle is repeated 3 times.

[0116] S3-S6 Same as Example 1.

[0117] Effect evaluation: 87.2 kg of yeast extract product was obtained (60% solids content, 52.3 kg on dry basis).

[0118] Dry basis yield = 52.3 kg / 100.0 kg × 100% = 52.3%.

[0119] The product has a dry-basis protein content of 55.8%.

[0120] The protein content entering the product = 52.3 kg × 55.8% = 29.18 kg.

[0121] Protein extraction rate = 29.18 kg / 52.0 kg × 100% = 56.1%.

[0122] Comparative Example 1: Conventional autolysis process The same batch of wet mycelium sludge raw material as in Example 1 was used (equivalent to 100.0 kg dry basis, total protein 52.0 kg).

[0123] The washed wet bacterial sludge was prepared into a suspension with a dry matter concentration of about 15% using purified water, and the pH was adjusted to 5.5 and the temperature was 50℃.

[0124] Add 1.5% sodium chloride (by dry weight of yeast) to promote autolysis, and autolysis is carried out for 24 hours with stirring.

[0125] After autolysis, the temperature was raised to 85°C for 30 minutes to inactivate the enzyme, and the supernatant was obtained by centrifugation without washing with water.

[0126] The supernatant was concentrated under the same vacuum concentration conditions as in Example 1 until the solid content was 60%.

[0127] Effect evaluation: 58.3 kg of yeast extract product was obtained (60% solids content, 35.0 kg on dry basis).

[0128] Dry basis yield = 35.0 kg / 100.0 kg × 100% = 35.0%.

[0129] The product has a dry-basis protein content of 46.5%.

[0130] The protein content entering the product = 35.0 kg × 46.5% = 16.3 kg.

[0131] Protein extraction rate = 16.3 kg / 52.0 kg × 100% = 31.3%.

[0132] Comparative Example 2: Synergistic cell disruption followed by mixed enzymatic hydrolysis process (not divided into steps) The same batch of wet bacterial sludge raw material as in Example 1 (equivalent to 100.0 kg dry basis, total protein 52.0 kg) was used. The cell wall breaking process was exactly the same as in Example 1.

[0133] After cell wall disruption, the yeast homogenate was adjusted to pH 6.8 and the temperature was 55℃ (water bath).

[0134] The compound cell-wall-breaking enzyme used in Example 1 (mass ratio of cell-wall-breaking enzyme: β-glucanase: mannanase = 2:0.8:0.8) and Bacillus subtilis neutral protease were added simultaneously. The amount of compound cell-wall-breaking enzyme added (by weight) was 0.25% of the dry weight of yeast; the amount of neutral protease added (by weight) was 0.45% of the dry weight of yeast. The total amount added was the same as the sum of the two enzymes in Example 1. The enzymatic hydrolysis reaction was carried out with stirring (60°C, 200 rpm) for 10 hours.

[0135] The subsequent inactivation, water extraction, solid-liquid separation (without water washing), and concentration processes were performed under the same conditions as in Example 1.

[0136] Effect evaluation: 75.0 kg of yeast extract product was obtained (60% solids content, 45.0 kg on dry basis).

[0137] Dry basis yield = 45.0 kg / 100.0 kg × 100% = 45.0%.

[0138] The product has a dry-basis protein content of 51.0%.

[0139] The protein content entering the product = 45.0 kg × 51.0% = 22.95 kg.

[0140] Protein extraction rate = 22.95 kg / 52.0 kg × 100% = 44.1%.

[0141] Comparative Example 3: Stepwise enzymatic hydrolysis but waterless extraction step The process steps S1-S3 are exactly the same as in Example 1, until the inactivation in step S4.

[0142] After inactivation, the water extraction step of "adding an equal mass of purified water and stirring at 40°C" is not performed; instead, centrifugation is performed directly.

[0143] The separated solid phase was washed only once with water (1 times the mass of the solid phase in water), and the supernatants were combined after centrifugation.

[0144] The concentration process is the same as in Example 1.

[0145] Effect evaluation: 83.3 kg of yeast extract product was obtained (60% solids content, 50.0 kg on dry basis).

[0146] Dry basis yield = 50.0 kg / 100.0 kg × 100% = 50.0%.

[0147] The product has a dry-basis protein content of 56.0%.

[0148] The protein content entering the product = 50.0 kg × 56.0% = 28.0 kg.

[0149] Protein extraction rate = 28.0 kg / 52.0 kg × 100% = 53.8%.

[0150] Comparative Example 4: Synergistic cell disruption + mixed enzymatic hydrolysis (unstepped) + water extraction Compared with Example 1, this comparative example only changed the enzymatic hydrolysis method from "stepwise enzymatic hydrolysis" to "mixed enzymatic hydrolysis (without steps)" (same as Comparative Example 2). All other conditions (including raw material preparation, suspension preparation and cell wall breaking treatment, inactivation and water extraction, solid-liquid separation and washing, and concentration) were exactly the same as in Example 1.

[0151] Effect evaluation: 86.5 kg of yeast extract product was obtained (60% solids content, 51.9 kg on dry basis).

[0152] Dry basis yield = 51.9 kg / 100.0 kg × 100% = 51.9%.

[0153] The product has a dry-basis protein content of 53.2%.

[0154] The protein content entering the product = 51.9 kg × 53.2% = 27.61 kg.

[0155] Protein extraction rate = 27.61 kg / 52.0 kg × 100% = 53.1%.

[0156] The results of the yeast extracts prepared in Examples 1-6 and Comparative Examples 1-4 are shown in Table 1.

[0157] Table 1. Results of yeast extracts prepared in Examples 1-6 and Comparative Examples 1-4 As shown in Table 1: The protein extraction rate of Examples 1-6 of this invention is 56.1%~65.0%, and the dry basis protein content of the product is 55.8%~59.5%. Both indicators are significantly better than those of Comparative Examples 1-4 (protein extraction rate 31.3%~53.8%, dry basis protein content of the product 46.5%~56.0%).

[0158] The comparison between Example 1 and Comparative Examples 2 and 4 shows that, under the same cell wall disruption conditions and total enzyme addition, the protein extraction rate of the stepwise enzymatic hydrolysis process (61.3%) is 8.2 to 17.2 percentage points higher than that of the mixed enzymatic hydrolysis process (44.1% to 53.1%), proving that the enzymatic hydrolysis sequence has a key technological contribution.

[0159] The comparison between Example 1 and Comparative Example 3 shows that the water extraction step after inactivation can increase the protein extraction rate by 7.5 percentage points. In Examples 2-6, the protein extraction rate remained above 55% despite changes in parameters such as enzyme addition, hydrolysis conditions, and homogenization pressure, indicating that the process of this invention has a wide operating window and good parameter adaptability.

[0160] Furthermore, although the protein content of Comparative Example 3 (56.0%) was comparable to that of Example 6 (55.8%), its extraction rate (53.8%) was significantly lower than that of Example 6 (56.1%), demonstrating the necessity and advanced nature of "protein extraction rate" as a core evaluation indicator.

[0161] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0162] Experiment 1: Screening Experiment for Compound Cell Wall Disrupting Enzymes 1. Using the same batch of wet mycelium sludge raw material as in Example 1 (dry basis protein content 52.0%), the following process conditions were kept constant: S2. Cell wall disruption treatment: Homogenization pressure 90 MPa, 2 cycles, the rest is the same as in Example 1; S3. Amount of neutral protease added in the second enzymatic hydrolysis of Bacillus subtilis: The amount of neutral protease added (by weight) of Bacillus subtilis is 0.45% of the dry weight of yeast. Enzymatic hydrolysis is carried out for 5 hours, and the rest is the same as in Example 1. S4 inactivation and water extraction, S5 solid-liquid separation and washing, and S6 concentration are exactly the same as in Example 1.

[0163] The only variable is the composition and ratio of the compound cell-wall breaking enzyme in the first enzymatic hydrolysis in S3. Other conditions for enzymatic hydrolysis are the same as in Example 1. The amount of compound cell-wall breaking enzyme added (by weight) is 0.25% of the dry weight of yeast.

[0164] As shown in Table 2, the following 7 treatment groups were set up, and each group was subjected to 3 parallel experiments, and the average value was taken.

[0165] Table 2. Grouping of the composite cell-wall-breaking enzyme screening experiment Note: The sources of glucanase, mannanase, and cell wall-breaking enzyme are the same as in Example 1.

[0166] 2. Evaluation indicators: (1) Soluble protein release after the first enzymatic hydrolysis: After the first enzymatic hydrolysis in S3 was completed, samples were taken immediately, centrifuged at 12000 rpm for 10 minutes at 4℃, and the supernatant was taken. The concentration of soluble protein (unit: mg / mL) was determined by the Bradford method. (2) Protein extraction rate of final product: As defined in this invention, it is calculated after the entire process is completed.

[0167] 3. Experimental Results: Table 3. Experimental Results 4. Results Analysis: 4.1 Comparison of Single Enzyme Effects The ability of a single enzyme to degrade the yeast cell wall is limited. Among them, the cell wall breaking enzyme (group 1) is more effective than glucanase (group 2) and mannanase (group 3). However, when the three enzymes are used alone, the concentration of soluble protein after the first enzymatic hydrolysis is less than 19 mg / mL, and the final protein extraction rate is less than 50%, which is significantly lower than that of the combined enzyme group.

[0168] 4.2 Effect of dual-enzyme combination Any combination of two enzymes (groups 4, 5, and 6) was superior to single enzymes, increasing the soluble protein concentration to 19-22 mg / mL and the protein extraction rate to 51%-55%. Among them, the combination of cell wall disrupting enzyme and dextranase (group 4) showed the best effect, but it was still significantly lower than the three-enzyme combination.

[0169] 4.3 Synergistic effect of three enzymes The three-enzyme combination (Group 7) achieved a soluble protein concentration of 26.8 mg / mL after the first enzymatic hydrolysis, which was 25.8% higher than the optimal two-enzyme combination (Group 4, 21.3 mg / mL); the final protein extraction rate reached 61.3%, which was 7.1 percentage points higher than the optimal two-enzyme combination (Group 4, 54.2%).

[0170] This invention employs a synergistic combination of three enzymes: cell wall-breaking enzyme, glucanase, and mannanase, which significantly improves the release of soluble protein and the final protein extraction rate. This experimental example fully demonstrates that the selection of components for the composite cell wall-breaking enzyme in this invention has a clear technical basis and a significant synergistic effect, and is not an arbitrary combination or empirical selection.

[0171] In summary, this invention provides a method for preparing high-protein yeast extract, which can effectively increase the protein content in yeast extract. Furthermore, this method features a short production cycle and mild, controllable conditions. The process is rational and suitable for industrialization, possessing promising prospects for commercialization.

Claims

1. A method for preparing yeast extract, characterized in that: Includes the following steps: S1. Centrifuge the yeast fermentation broth to obtain wet bacterial sludge; S2. Mix the wet bacterial mud with water to form a suspension, and then homogenize and break down the cell walls of the suspension to obtain a slurry; S3. Add compound cell wall-breaking enzyme to the slurry for enzymatic hydrolysis, and then add neutral protease for enzymatic hydrolysis; S4. After enzymatic hydrolysis, the enzyme is inactivated and then water is added for extraction; S5. Perform solid-liquid separation on the extracted material and collect the supernatant; S6. Concentrate the obtained supernatant to obtain yeast extract.

2. The method for preparing yeast extract according to claim 1, characterized in that: In step S1, the wet bacterial sludge is washed with water after centrifugation and then centrifuged again. And / or, in step S2, the mass ratio of the wet bacterial mud to water is (6-8):(4-2); And / or, in step S2, the pressure of the homogenization is 60-120 MPa.

3. The method for preparing yeast extract according to claim 2, characterized in that: In step S1, the centrifugation speed is 6000-8000 rpm, and the centrifugation time is 10-30 minutes; And / or, in step S2, the homogenization pressure is 80-100 MPa, and the homogenization is performed 1-3 times.

4. The method for preparing yeast extract according to claim 1, characterized in that: In step S3, before adding the compound cell wall-breaking enzyme, the temperature of the slurry is adjusted to 55-65℃ and the pH is adjusted to 5.0-6.0; And / or, in step S3, the composite cell-wall-breaking enzyme is composed of cell-wall-breaking enzyme, dextranase, and mannanase in a mass ratio of (1-3):(0.5-1):(0.5-1); And / or, in step S3, the amount of the compound cell-wall-breaking enzyme added is 0.2-0.4% of the dry weight of yeast; And / or, in step S3, the addition of the compound cell-wall-breaking enzyme for enzymatic hydrolysis is carried out by stirring at 60-65℃ and 100-500rpm for 3-4 hours.

5. The method for preparing yeast extract according to claim 4, characterized in that: In step S3, the mass ratio of the cell wall breaking enzyme, glucanase, and mannanase is 2:0.8:0.

8.

6. The method for preparing yeast extract according to claim 1, characterized in that: In step S3, the neutral protease is Bacillus subtilis neutral protease; And / or, in step S3, the amount of neutral protease added is 0.4-0.6% of the dry weight of yeast; And / or, in step S3, the addition of neutral protease for enzymatic hydrolysis is performed by stirring at pH 5.0-6.0, 60-65℃, and 100-500 rpm for 4-6 hours.

7. The method for preparing yeast extract according to claim 1, characterized in that: In step S4, the inactivation is performed by incubating at 80-95°C for 10-30 minutes to inactivate the enzyme; And / or, in step S4, the volume of the water is 1-2 times the volume of the inactivated enzymatic hydrolysate; And / or, in step S4, the extraction is performed by stirring at 35-45°C for 0.5-1 hour; And / or, in step S6, the concentration is carried out under vacuum conditions of -0.080 to -0.090 MPa and 55-65°C until the solid content reaches 55%-65%.

8. The method for preparing yeast extract according to claim 7, characterized in that: In step S4, before inactivation, the system is adjusted to a temperature of 45-55℃ and a pH of 6.5-7.5, and flavor protease is added for treatment for 1-2 hours. The amount of flavor protease added is 0.05%-0.2% of the dry weight of yeast.

9. A yeast extract, characterized in that: It is prepared by the yeast extract preparation method according to any one of claims 1 to 8.

10. The use of the yeast extract according to claim 9 in the fields of food flavoring, fermentation culture medium, and biological fermentation aid.

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