Method for recovering all components of beer waste yeast

Abstract

The present application relates to a kind of beer waste yeast full component recovery method, belong to waste yeast comprehensive recycling field.To the yeast suspension prepared from waste yeast, add composite enzyme for enzymolysis, after cooling, utilize rotary membrane separation, residual yeast particles are obtained by precipitating, drying and crushing;Large molecular extract and small molecular extract are obtained by ultrafiltration of flow-through fluid.After the large molecular extract is concentrated, add (NH4) 2SO4 to precipitate protein, centrifugal, redissolve precipitate, obtain yeast protein by utilizing rotary membrane separation;After desalination of flow-through fluid by dialysis, add anhydrous ethanol to precipitate, after centrifugation, redissolve precipitate, obtain yeast polysaccharide by freeze-drying.Small molecular extract is enriched by macroporous resin, and yeast small molecular extract is obtained by freeze-drying after elution.The wastewater generated in the whole process is regenerated into industrial water that can be directly reused by using integrated process of physical and chemical pretreatment and advanced denitrification and phosphorus removal.The present application has the advantages of low cost, high extraction efficiency, green environmental protection and realizing waste yeast full component recovery, resource closed loop, etc.

Description

Technical Field

[0001] This invention relates to the field of comprehensive recycling of waste yeast, and in particular to a method for the complete recovery of all components of waste beer yeast. Background Technology

[0002] Waste brewer's yeast is a byproduct generated during beer production when brewer's yeast proliferates and ferments in wort. After fermentation, its activity level is lower than required for beer production or its yield exceeds the brewery's normal recycling capacity. Every 1000 tons of beer produced generates 1-1.5 tons of waste yeast. my country is the world's largest beer producer and consumer, consistently ranking first in annual production, resulting in a massive quantity of yeast sludge—a byproduct of beer brewing.

[0003] Brewer's yeast is rich in components, containing approximately 50% protein on a dry basis, including all eight essential amino acids, and 6-8% RNA. The cell walls of yeast cells contain 25-35% yeast polysaccharides, mainly glucan and mannan. Brewer's yeast is also rich in vitamins and minerals, particularly vitamins B1, B2, B6, and B6. 12 Furthermore, yeast sludge exists primarily in the form of phosphate esters, making it easily absorbed by the human body. In addition, yeast cells contain abundant enzyme systems and physiologically active substances, such as coenzyme A, coenzyme Q, coenzyme I, cytochrome C, glutathione, and ergosterol. However, currently, most beer companies only use it directly or after simple drying as a feed additive, resulting in low added value and limited economic benefits. A large amount of yeast sludge is discarded without in-depth treatment, wasting biological resources and failing to explore its application potential in high-value fields such as food flavoring, nutrition and health care, and biomedicine. This also brings significant environmental pressure. If not properly disposed of, waste yeast sludge easily decomposes and ferments in the natural environment, producing foul-smelling gases and potentially leaching high concentrations of organic matter and nitrogen- and phosphorus-containing wastewater, polluting water bodies and soil, and increasing the environmental governance costs for enterprises.

[0004] While existing technologies can extract single components, such as yeast extracts, they fail to systematically achieve the synergistic separation of multiple components, making it difficult to balance recovery rate, product purity, and economic efficiency. Therefore, developing a process capable of efficiently separating different components is of great significance for enhancing the comprehensive utilization value of waste brewer's yeast and alleviating environmental pressure. Summary of the Invention

[0005] To address the issues of low extraction efficiency and limited component recovery in existing waste yeast recycling methods, this invention provides a method for the complete recovery of all components from waste brewer's yeast. This process, through systematic step design, aims to achieve efficient separation of proteins, polysaccharides, small molecule active substances, and residual solids from waste brewer's yeast, thereby maximizing resource utilization and economic benefits.

[0006] This invention is achieved through the following technical solution:

[0007] The purpose of this invention is to provide a method for the complete recovery of waste brewer's yeast, comprising the following steps:

[0008] (1) Mix the waste brewer's yeast with the compound enzyme in a buffer solution and hydrolyze to obtain the hydrolysate;

[0009] (2) After cooling the enzymatic hydrolysate obtained in step (1), spin membrane separation was performed to obtain flow-through liquid 1 and precipitate; the precipitate was dried and pulverized to obtain residual yeast particles;

[0010] (3) Ultrafiltration of the flow-through liquid 1 obtained in step (2) yields retentate and flow-through liquid 2;

[0011] (4) Salt out the protein precipitated from the retentate obtained in step (3), and then separate it with a spinning membrane to obtain yeast protein and flow-through liquid 3;

[0012] (5) The flow-through liquid 3 obtained in step (4) was desalted by dialysis, precipitated with anhydrous ethanol, and after solid-liquid separation, the obtained solid was re-dissolved and freeze-dried to obtain yeast polysaccharide.

[0013] (6) The flow-through liquid 2 obtained in step (3) is adsorbed by macroporous resin to obtain unadsorbed flow-through liquid 4; the adsorbed macroporous resin is eluted, the eluent is concentrated and freeze-dried to obtain yeast small molecule extract.

[0014] Wastewater recovery treatment is carried out on the flow-through liquid 4.

[0015] In one embodiment of the present invention, in step (1), the complex enzyme is mannanase, dextranase and hemicellulase.

[0016] In one embodiment of the present invention, in step (1), the mass ratio of mannanase, dextranase and hemicellulase added is 5-10:12-18:5-10.

[0017] In one embodiment of the present invention, in step (1), the mass ratio of the waste beer yeast to the compound enzyme is 100:0.2-0.5.

[0018] In one embodiment of the present invention, in step (1), the buffer solution is sodium acetate-acetic acid buffer solution, sodium citrate-citric acid buffer solution or ammonium acetate-acetic acid solution;

[0019] And / or, the pH of the buffer solution is 4.5-6.0.

[0020] In one embodiment of the present invention, in step (1), the enzymatic hydrolysis conditions are: 30-50℃ for 5-7 h, preferably 40-45℃ for 6-7 h.

[0021] In one embodiment of the present invention, in step (2), the separation temperature is 2℃-6℃.

[0022] In one embodiment of the present invention, in step (3), the molecular weight cutoff of the ultrafiltration is 10 kDa.

[0023] In one embodiment of the present invention, in step (4), the precipitated protein is: protein precipitated by (NH4)2SO4 with a saturation of 60%-80% under conditions of 2℃-6℃.

[0024] In one embodiment of the present invention, in step (5), the precipitation is carried out at 2-6°C.

[0025] In one embodiment of the present invention, in step (5), the molecular weight of the yeast polysaccharide is 20 kDa-60 kDa.

[0026] In one embodiment of the present invention, in step (6), the macroporous adsorption resin is a weakly polar or non-polar styrene-based macroporous adsorption resin; the macroporous resin is selected from AB-8 macroporous resin, HPD-300 macroporous resin, HPD-100 macroporous resin or D101 macroporous resin.

[0027] In one embodiment of the present invention, in step (6), the wastewater recycling treatment includes one or more of the following processes: physicochemical pretreatment, anaerobic resource recovery, deep nitrogen and phosphorus removal, and MBR membrane reuse.

[0028] The flow-through solution 4 mainly contains ions such as phosphorus, ammonia nitrogen, potassium, sodium, calcium, and magnesium, with pH=4.98, conductivity of 677 μS / cm, and COD of 8600 mg / L.

[0029] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0030] (1) This invention uses an enzymatic hydrolysis-membrane separation process to systematically recover proteins, polysaccharides and small molecule active substances from waste yeast. The resource utilization rate is close to 100%, and it has the potential to fully transform low-value by-products into high-value-added products for high-end applications, which is expected to improve economic benefits.

[0031] (2) This invention organically couples modern technologies such as enzyme engineering, membrane separation and resin adsorption to achieve precise and efficient separation of multiple components. The process end integrates a wastewater treatment and reuse system, forming a clean production closed loop from waste to diversified products with near-zero wastewater discharge.

[0032] (3) Compared with the prior art, the present invention has significant advantages in terms of enzymatic hydrolysis efficiency, resource utilization, product diversity, yield, purity and technical transparency: the existing process only uses a single protease or a simple complex enzyme, which does not fully degrade the yeast cell wall and focuses on the extraction of a single product, resulting in low resource utilization and easy waste of by-products; while the present invention clearly discloses a complex enzyme system composed of mannanase, glucanase and hemicellulase, which can specifically degrade the polysaccharide structure of yeast cell wall, realize the full-component recovery of yeast cells, yeast polysaccharides, yeast small molecules, yeast proteins and wastewater, and form a complete resource recycling loop, which maximizes the raw material value of beer waste yeast and conforms to the concept of green production and circular economy; in addition, the enzymatic hydrolysis conditions of the present invention are relatively mild (pH 4.5–6, temperature 30–50℃), which can effectively avoid the destruction of product activity by high temperature and extreme pH, and is more conducive to preserving the natural functional characteristics of polysaccharides, proteins and other products, providing support for the expansion of downstream high value-added applications, and achieving a synergistic improvement in production efficiency, resource utilization and environmental benefits. Attached Figure Description

[0033] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0034] Figure 1 This is a process flow diagram of the present invention;

[0035] Figure 2 The time conditions for the compound enzymatic yeast autolysis of this invention were optimized.

[0036] Figure 3 These are the results of the monosaccharide composition analysis of yeast polysaccharides in this invention;

[0037] Figure 4 This invention relates to the effect of yeast polysaccharides on oxidative stress in zebrafish; wherein, A represents zebrafish morphology and fluorescence images; B represents survival rate; and C represents quantitative fluorescence intensity.

[0038] Figure 5 The whitening effect of the yeast small molecule extract of this invention was tested.

[0039] Figure 6 This is a flow chart of the wastewater recycling and treatment process of the present invention. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0042] The waste brewer's yeast liquid comes from China Resources Snow Breweries (China) Co., Ltd.

[0043] Mannanase, glucanase, and hemicellulase all originated from Ningxia Xiasheng Industrial Group Co., Ltd., with production batch number 22507016.

[0044] Example 1

[0045] This embodiment provides an integrated process for the complete recovery and high-value conversion of waste brewer's yeast (flowchart shown below). Figure 1 (As shown). The process includes the following steps:

[0046] (1) Weigh 400 g of waste brewer's yeast, add compound enzyme (of which mannanase, glucanase and hemicellulase account for 10%, 15% and 8% of the total mass of compound enzyme, respectively) at 0.5% of the weight of waste brewer's yeast, mix in 4 L sodium acetate-acetic acid buffer solution (concentration of 0.2 M) at pH=5.2, heat and stir at 40℃ for 6 h to obtain enzymatic hydrolysate;

[0047] (2) After cooling the enzymatic hydrolysate obtained in step (1), separate it using a rotating membrane to obtain flow-through liquid 1 and precipitate; collect the precipitate, dry and pulverize it to obtain residual yeast particles;

[0048] (3) The flow-through liquid 1 obtained in step (2) is ultrafiltered with a hollow fiber column with a molecular weight cutoff of 10 kDa to obtain a retentate (macromolecule extract) and a flow-through liquid 2 (small molecule extract).

[0049] (4) Add the retentate (macromolecule extract) obtained in step (3) to (NH4)2SO4 with a saturation of 80%, precipitate the protein at 4 ℃, let stand overnight, and use a rotating membrane to separate the resulting turbid liquid to obtain yeast protein and flow-through liquid 3.

[0050] (5) After dialysis to remove salt from the flow-through liquid 3 obtained in step (4), add four times the volume of anhydrous ethanol to precipitate it. After standing overnight, centrifuge at 4°C and 4500 rpm for 15 min. The precipitate is re-dissolved in pure water, and the residual ethanol is evaporated in a boiling water bath to obtain a polysaccharide solution. The solution is then freeze-dried to obtain yeast polysaccharide.

[0051] (6) The flow-through liquid 2 (small molecule extract) in step (3) is adsorbed and enriched with AB-8 macroporous resin. The macroporous resin adsorbing the target small molecules is eluted with ethanol. The ethanol eluent is concentrated and freeze-dried to obtain yeast small molecule extract.

[0052] After loading the macroporous adsorption resin, the small molecule solution (flow-through solution 4) that was not adsorbed by the resin was recovered through an integrated process of physicochemical pretreatment, anaerobic resource recovery, deep nitrogen and phosphorus removal, and MBR membrane reuse (see the detailed flow chart). Figure 6 This refers to industrial water that can be used directly.

[0053] Example 2

[0054] This embodiment provides an integrated process for the complete recovery and high-value conversion of waste brewer's yeast. The process includes the following steps:

[0055] (1) Weigh 400 g of waste beer yeast, add compound enzyme (of which mannanase, glucanase and hemicellulase account for 5%, 12% and 10% of the total mass of compound enzyme, respectively) at 0.5% of the weight of waste beer yeast, mix in 4 L sodium acetate-acetic acid buffer solution (concentration of 0.2 M) at pH=5.2, heat and stir at 30℃ for 6 h to obtain enzymatic hydrolysate;

[0056] (2) After cooling the enzymatic hydrolysate obtained in step (1), separate it using a rotating membrane to obtain flow-through liquid 1 and precipitate; collect the precipitate, dry and pulverize it to obtain residual yeast particles;

[0057] (3) The flow-through liquid 1 obtained in step (2) is ultrafiltered with a hollow fiber column with a molecular weight cutoff of 10 kDa to obtain a retentate (macromolecule extract) and a flow-through liquid 2 (small molecule extract).

[0058] (4) Add the retentate (macromolecule extract) obtained in step (3) to (NH4)2SO4 with a saturation of 80%, precipitate the protein at 4 ℃, let stand overnight, and use a rotating membrane to separate the resulting turbid liquid to obtain yeast protein and flow-through liquid 3.

[0059] (5) After dialysis to remove salt from the flow-through liquid 3 obtained in step (4), add four times the volume of anhydrous ethanol to precipitate it. After standing overnight, centrifuge at 4°C and 4500 rpm for 15 min. The precipitate is re-dissolved in pure water, and the residual ethanol is evaporated in a boiling water bath to obtain a polysaccharide solution. The solution is then freeze-dried to obtain yeast polysaccharide.

[0060] (6) The flow-through liquid 2 (small molecule extract) in step (3) is adsorbed and enriched by AB-8 macroporous resin, and the ethanol eluent is concentrated and freeze-dried to obtain yeast small molecule extract.

[0061] After the macroporous adsorption resin is loaded, the small molecule solution that is not adsorbed by the resin (flow-through liquid 4) is recycled into directly usable industrial water through a physicochemical pretreatment, anaerobic resource recovery, deep denitrification and phosphorus removal, and MBR membrane reuse integrated process.

[0062] Example 3

[0063] This embodiment provides an integrated process for the complete recovery and high-value conversion of waste brewer's yeast. The process includes the following steps:

[0064] (1) Weigh 400 g of waste brewer's yeast, add compound enzyme (of which mannanase, glucanase and hemicellulase account for 10%, 18% and 5% of the total mass of compound enzyme, respectively) at 0.5% of the weight of waste brewer's yeast, mix in 4 L sodium acetate-acetic acid buffer solution (concentration of 0.2 M) at pH=5.2, heat and stir at 50℃ for 6 h to obtain enzymatic hydrolysate;

[0065] (2) After cooling the enzymatic hydrolysate obtained in step (1), separate it using a rotating membrane to obtain flow-through liquid 1 and precipitate; collect the precipitate, dry and pulverize it to obtain residual yeast particles;

[0066] (3) The flow-through liquid 1 obtained in step (2) is ultrafiltered with a hollow fiber column with a molecular weight cutoff of 10 kDa to obtain a retentate (macromolecule extract) and a flow-through liquid 2 (small molecule extract).

[0067] (4) Add the retentate (macromolecule extract) obtained in step (3) to (NH4)2SO4 with a saturation of 80%, precipitate the protein at 4 ℃, let stand overnight, and use a rotating membrane to separate the resulting turbid liquid to obtain yeast protein and flow-through liquid 3.

[0068] (5) After dialysis to remove salt from the flow-through liquid 3 obtained in step (4), add four times the volume of anhydrous ethanol to precipitate it. After standing overnight, centrifuge at 4°C and 4500 rpm for 15 min. The precipitate is re-dissolved in pure water, and the residual ethanol is evaporated in a boiling water bath to obtain a polysaccharide solution. The solution is then freeze-dried to obtain yeast polysaccharide.

[0069] (6) The flow-through liquid 2 (small molecule extract) in step (3) is adsorbed and enriched by AB-8 macroporous resin, and the ethanol eluent is concentrated and freeze-dried to obtain yeast small molecule extract.

[0070] After the macroporous adsorption resin is loaded, the small molecule solution that is not adsorbed by the resin (flow-through liquid 4) is recycled into directly usable industrial water through a physicochemical pretreatment, anaerobic resource recovery, deep denitrification and phosphorus removal, and MBR membrane reuse integrated process.

[0071] Test Example 1: Investigation of Compound Enzymatic Hydrolysis Time

[0072] To investigate the effect of enzymatic autolysis time on extraction efficiency, the total sugar content in the extract was used as an indicator. The total sugar content at each time point was evaluated using the phenol-sulfuric acid method to optimize the yeast extraction process. The specific experiments are as follows:

[0073] Take 30 mL of waste brewer's yeast liquid and place it in a 1 L beaker. Prepare a sodium acetate-acetic acid buffer solution with pH=5.2. The ratio of yeast liquid to buffer solution is 1:10 (v / v). First, add 200 mL of buffer solution and mix well. Using the weight of yeast liquid as a reference, weigh 0.5% of the complex enzyme (mannanase, glucanase, and hemicellulase account for 10%, 15%, and 8% of the total mass of the complex enzyme, respectively). Dissolve it in 100 mL of buffer solution, add it to the beaker, mix well, and stir at 500 rpm at 40 ℃ for 8 h. Detect the sugar content of the solution after diluting it tenfold using the phenol-sulfuric acid method.

[0074] The results are as follows Figure 2 As shown, the total sugar content in the extract first increased and then decreased over time, reaching a peak at 6 h. Therefore, the process conditions for this compound enzymatic yeast autolysis were determined as follows: temperature 40 ℃, enzyme dosage 0.5%, buffer system of sodium acetate-acetic acid buffer solution with pH=5.2, and enzymatic time 6 h.

[0075] Test Example 2: Monosaccharide Composition Analysis of Yeast Polysaccharides

[0076] The monosaccharide composition of the yeast polysaccharide prepared in Example 1 was determined by ion exchange chromatography (IC). An IC system (IC Professional 850, Metrohm China Co. Ltd.) equipped with a pulsed amperometric detector was used. The column was a Carbopac PA1 (4.6 × 250 mm, ThermoFisher, USA). The mobile phase consisted of 15 mM sodium hydroxide solution and a mixture of 150 mM sodium acetate and 15 mM sodium hydroxide. The flow rate was 1 mL / min, and the column temperature was 30 °C. Each injection consisted of 20 μL of standard or sample. Sample preparation followed this procedure: 3 mg of yeast polysaccharide was dissolved in 0.6 mL of ultrapure water, and an equal volume of 2 M trifluoroacetic acid solution was added. The mixture was placed in a 100 °C oven for acid hydrolysis for 12 h. The pH was then adjusted to neutral with 1 M sodium hydroxide solution, and the volume was brought to 5 mL with water. The sample was filtered through a 0.22 μm aqueous membrane before injection.

[0077] Qualitative and quantitative analysis of yeast polysaccharides was performed by comparing the retention times and standard curves of different monosaccharide standards. The chromatographic separation results of yeast polysaccharides are shown below. Figure 3 As shown. The yeast polysaccharides primarily contain mannose and glucose, with a mass ratio of rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid, and glucuronic acid of 1.89:2.97:9.65:11.32:69.08:3.84:1.25. Therefore, the yeast polysaccharide extracted in Example 1 is glucomannan.

[0078] Test Example 3: Effects of yeast polysaccharides on oxidative stress in zebrafish

[0079] The yeast polysaccharide prepared in Example 1 was used to prepare solutions of 50 μg / mL, 100 μg / mL, and 200 μg / mL using zebrafish culture medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2·2H2O, 0.33 mM MgSO4·7H2O, 0.5 mg / L methylene blue) as solvent. The experiment was divided into 5 groups: a blank control group, a model group, and polysaccharide experimental groups (50 μg / mL, 100 μg / mL, and 200 μg / mL). Blank control group: 24 hpf zebrafish embryos were exposed only to the culture water. Model group: 24 hpf zebrafish embryos were added to 6-well plates containing 3 mL of culture water, cultured for 12 h, and then incubated with 5 mM H2O2 solution for 36 h under dark conditions. Polysaccharide Experimental Group: Zebrafish embryos at 24 hpf were added to 6-well plates containing 3 mL of culture water. The embryos were first treated with different concentrations (50 μg / mL, 100 μg / mL, and 200 μg / mL) of crude yeast polysaccharide solution for 12 hours. After pretreatment, 5 mM H2O2 solution was added, and the embryos were incubated in the dark for 36 hours. All experiments had three replicates, with n = 20 samples per group. After culture, zebrafish morphology was recorded under a microscope to observe for deformities and survival rates. In vivo ROS levels were then measured to reflect the oxidative stress level of the zebrafish. The reactive oxygen species (ROS) level was detected using the fluorescent dye 2,7-dichlorofluorescein diacetate (DCFH-DA). The specific procedure was as follows: the hatched larvae were transferred to 24-well plates, and 1 mL of 20 μg / mL DCFH-DA solution was added and incubated in the dark for 1 hour. After rinsing twice with culture water, the larvae were anesthetized with MS-222, photographed, and quantitatively analyzed.

[0080] The results are as follows Figure 4 As shown, after cultivation, the morphology of zebrafish larvae in each group was observed under a microscope. Compared with the blank control, the model group showed obvious deformities and a significantly lower survival rate. The experimental group larvae did not show obvious deformities, but the low-dose group larvae showed some degree of damage to the heart, manifesting as reddening of the heart. Regarding the determination of ROS levels in zebrafish, the figure shows that the ROS level in the model group was significantly increased, with higher fluorescence intensity, indicating that hydrogen peroxide significantly increased oxidative stress levels in zebrafish. Compared with the model group, the fluorescence intensity of each experimental group was lower, while the fluorescence intensity of the high-dose group was similar to that of the blank group. Therefore, yeast polysaccharides can inhibit the increase in oxidative stress levels in zebrafish induced by hydrogen peroxide.

[0081] Oxidative stress is a significant factor contributing to aging and various chronic diseases. Therefore, ingredients with antioxidant properties have attracted considerable attention in the field of functional foods. The yeast polysaccharide obtained in this invention has been shown to have significant antioxidant activity in a zebrafish model, providing a solid scientific basis for its development as a core ingredient in functional foods with anti-aging and immune-enhancing properties.

[0082] Test Example 4: Whitening Experiment of Yeast Small Molecule Extract

[0083] The yeast small molecule extract prepared in Example 1 was used as a solvent in zebrafish culture medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2·2H2O, 0.33 mM MgSO4·7H2O, 0.5 mg / L methylene blue) to prepare solutions of 0.1 mg / mL and 0.2 mg / mL. The skin whitening experiment was divided into three groups: a blank control group and yeast small molecule extract experimental groups (0.1 mg / mL and 0.2 mg / mL). Sixty 8 hpf zebrafish embryos were added to 6-well plates containing 3 mL of culture water. The blank group was cultured in normal zebrafish culture medium. The experimental groups were treated with different concentrations (0.1 mg / mL and 0.2 mg / mL) of yeast small molecule solution, and the embryos were cultured to 3 dpf. After treatment, the morphology of the zebrafish was recorded under a microscope, and the distribution of melanin in each group was observed. Subsequently, the melanin content and tyrosinase activity in the zebrafish were measured. The specific procedures are as follows: For the determination of tyrosinase activity: After 72 h, the fish embryos were washed twice with culture water, and 5 mg / mL sodium deoxycholate solution was added. After ultrasonic disruption of the fish embryos, they were centrifuged for 5 min. 100 μL of the supernatant from each concentration group and 100 μL of levodopa solution (5 mmol / L) were added to a 96-well plate and mixed well. 100 μL of sodium deoxycholate solution was used as a blank control group instead of the supernatant. The plate was incubated at 37℃ for 1 h, and the absorbance at 475 nm was measured.

[0084] For melanin determination: Discard the supernatant remaining after centrifugation, add 300 μL of 1 mM sodium hydroxide and mix well to completely dissolve the precipitate. Take 100 μL of the solution into a 96-well plate, and take 100 μL of 1 mM sodium hydroxide as a blank control group, and measure the absorbance value at 405 nm. The melanin content of the blank group is taken as 100%.

[0085] For the two dose groups in the experimental group, from Figure 5It can be seen that the yeast small molecule extract has a significant whitening effect. The control group showed larger and more aggregated melanin patches, while the experimental group showed a significant reduction in melanin in the zebrafish heads, with no obvious melanin aggregation and smaller spots. The results of zebrafish tyrosinase activity and melanin content measurements showed that, compared with the control group, the experimental group exhibited reduced tyrosinase activity and significantly lower melanin content. Therefore, the small molecule extract can reduce melanin production in zebrafish, demonstrating a certain whitening effect. This indicates that the extracted yeast small molecules have great application potential in the field of cosmetic whitening.

[0086] Test Example 5: Compositional Analysis of Yeast Small Molecule Extract

[0087] The yeast small molecule extract prepared in Example 1 was used as a solvent to prepare a 10 mg / mL solution, which was then filtered through a 0.22 μm filter membrane before injection. A C18 column (250 × 4.6 mm, 5 μm) was used at a column temperature of 25 °C. A gradient elution program was employed with 0.1% formic acid aqueous solution (A) and acetonitrile (B) as the mobile phase, at a flow rate of 1 mL / min. Mass spectrometry was performed using an electrospray ionization (ESI) source in full-scan mode (m / z 50-1800), with a capillary voltage of 3.0 kV, an ion source temperature of 150 °C, a desolvation gas temperature of 400 °C, a cone gas flow rate of 50 L / h, and a desolvation gas flow rate of 800 L / h. Qualitative analysis was performed by comparing retention times and characteristic ion fragments ([M+H]⁺ / [MH]⁻, etc.).

[0088] HPLC analysis of the small molecule extract revealed three main peaks, representing the three components of the yeast small molecule extract. To further analyze the compound composition of each component, LC-MS / MS analysis was performed to preliminarily identify the major small molecule components. Fragment ion analysis and mass spectrometry database comparison identified seven key compounds from these three components, including glutathione, ergosterol, chlorogenic acid, citric acid, oleic acid, UDP-glucose, and uridine.

[0089] Test Example 6: Wastewater Recycling Process Development

[0090] The flow-through solution 4 obtained in Example 1 was sent to the Water and Environmental Technology R&D Center of Yancheng Environmental Protection Science and Technology City, Jiangsu Province for testing and wastewater recycling treatment. The flow-through solution 4 was found to contain phosphorus, ammonia nitrogen, potassium, sodium, calcium, and magnesium ions, with a pH of 4.98, a conductivity of 677 μS / cm, and a COD of 8600 mg / L. An integrated process was employed, including physicochemical pretreatment, anaerobic resource recovery, deep nitrogen and phosphorus removal, and MBR membrane reuse (see the detailed flow chart). Figure 6 The final water quality was pH 6-9, total phosphorus 0.4 mg / L, and COD 45 mg / L.

[0091] In this embodiment, pH is first adjusted and suspended solids are removed by air flotation; an anaerobic tower efficiently degrades organic matter (COD removal rate of 88%) and produces biogas; then nitrogen and phosphorus removal are enhanced by A2O process (anaerobic-anoxic-aerobic); finally, solid-liquid separation is achieved by MBR membrane to produce clean water that can be directly reused (COD ≤45 mg / L, total phosphorus ≤0.4 mg / L), forming a resource recovery closed loop, and the overall reuse cost can be controlled at 8~15 yuan / ton.

[0092] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for the complete recovery of all components from waste brewer's yeast, characterized in that, Includes the following steps: (1) Mix the waste brewer's yeast with the compound enzyme in a buffer solution and hydrolyze to obtain the hydrolysate; (2) After cooling the enzymatic hydrolysate obtained in step (1), spin membrane separation was performed to obtain flow-through liquid 1 and precipitate; the precipitate was dried and pulverized to obtain residual yeast particles; (3) Ultrafiltration of the flow-through liquid 1 obtained in step (2) yields retentate and flow-through liquid 2; (4) Salt out the protein precipitated from the retentate obtained in step (3), and then separate it with a spinning membrane to obtain yeast protein and flow-through liquid 3; (5) The flow-through liquid 3 obtained in step (4) was desalted by dialysis, precipitated with anhydrous ethanol, and after solid-liquid separation, the obtained solid was re-dissolved and freeze-dried to obtain yeast polysaccharide. (6) The flow-through liquid 2 obtained in step (3) is adsorbed by macroporous resin to obtain unadsorbed flow-through liquid 4; the adsorbed macroporous resin is eluted, the eluent is concentrated and freeze-dried to obtain yeast small molecule extract. Wastewater recovery treatment is carried out on the flow-through liquid 4.

2. The method according to claim 1, characterized in that, In step (1), the complex enzyme is mannanase, glucanase and hemicellulase.

3. The method according to claim 2, characterized in that, In step (1), the mass ratio of mannanase, glucanase and hemicellulase added is 5-10:12-18:5-10.

4. The method according to claim 1, characterized in that, In step (1), the mass ratio of the waste beer yeast to the compound enzyme is 100:0.2-0.

5.

5. The method according to claim 1, characterized in that, In step (1), the buffer solution is sodium acetate-acetic acid buffer solution, sodium citrate-citric acid buffer solution, or ammonium acetate-acetic acid solution; And / or, the pH of the buffer solution is 4.5-6.

0.

6. The method according to claim 1, characterized in that, In step (1), the enzymatic hydrolysis conditions are: 30-50℃ for 5-7 h, preferably 40-45℃ for 6-7 h.

7. The method according to claim 1, characterized in that, In step (2), the separation temperature is 2℃-6℃.

8. The method according to claim 1, characterized in that, In step (5), the molecular weight of the yeast polysaccharide is 20 kDa-60 kDa.

9. The method according to claim 1, characterized in that, In step (6), the macroporous adsorption resin is a weakly polar or non-polar styrene-based macroporous adsorption resin; the macroporous resin is selected from AB-8 macroporous resin, HPD-300 macroporous resin, HPD-100 macroporous resin or D101 macroporous resin.

10. The method according to claim 1, characterized in that, In step (6), the wastewater recycling treatment includes one or more of the following processes: physicochemical pretreatment, anaerobic resource recovery, deep nitrogen and phosphorus removal, and MBR membrane reuse.

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