Comprehensive utilization method of waste beer yeast

Abstract

The present invention proposes a comprehensive utilization method for waste beer yeast, which belongs to the field of comprehensive recycling of waste yeast and can solve the problems of large-scale yeast loss and acid-base reagent residue in existing waste yeast recycling methods. It mainly includes: adding compound enzyme A to a yeast solution prepared from waste yeast for enzymatic hydrolysis once, and centrifuging to obtain a supernatant and a precipitate; after the precipitate obtained from the first enzymatic hydrolysis is redissolved, it is subjected to secondary and tertiary enzymatic hydrolysis using compound enzyme B, and centrifuged to obtain a supernatant and a precipitate; after the precipitate obtained from the tertiary enzymatic hydrolysis is redissolved, it is subjected to shaking extraction in a boiling water bath, and centrifuged to obtain a yeast β-glucan precipitate and a supernatant containing yeast polysaccharide; the yeast β-glucan precipitate is subjected to NaOH boiling water extraction and four enzymatic hydrolysis, and the supernatant obtained by centrifugation is freeze-dried to obtain water-soluble yeast β-glucan; the supernatant containing yeast polysaccharide is freeze-dried to obtain a yeast polysaccharide additive. The present invention has the characteristics of simple operation, high extract recovery rate and the ability to achieve zero loss of waste yeast.

Description

Technical Field

[0001] The invention belongs to the field of comprehensive recycling of waste beer yeast, and in particular relates to a comprehensive utilization method of waste beer yeast. Background Art

[0002] Waste brewer's yeast is a byproduct of the beer industry, produced during the primary and secondary fermentation processes of beer production. Brewer's yeast is rich in nutrients. According to measurements, it contains approximately 50% protein, including eight essential amino acids, and 6-8% RNA. The yeast cell wall contains 25-35% yeast polysaccharides, primarily glucans and mannans. Brewer's yeast is rich in vitamins and minerals, particularly vitamins Bl, B2, B6, and Bl2. These nutrients are mostly present in the form of phosphate esters, making them easily absorbed by the body. Yeast cells also contain a rich array of enzymes and physiologically active substances, such as coenzyme A, coenzyme Q, coenzyme I, cytochrome C, coagulants, and glutathione.

[0003] In recent years, with the rapid development of my country's beer industry, beer production has increased year by year. According to statistics, my country's beer production reached 35.6243 million tons in 2021. However, every 100 tons of beer produced produces 1.5 tons of waste yeast sludge with a moisture content of 75% to 80%. It is calculated that the surplus yeast sludge produced in 2021 alone reached more than 534,400 tons. Currently, there are two main ways to deal with this waste yeast: drying it as animal feed or directly discharging it into the sewer. However, these methods not only waste biological resources but also pollute the environment. Therefore, how to achieve the comprehensive recycling and utilization of waste beer yeast is the key to solving the above-mentioned problems.

[0004] Existing methods for comprehensive recycling of waste yeast mostly use acid-base cleaning technology. However, this method not only causes a large loss of yeast, but also easily causes environmental pollution and acid-base reagent residues in the extract due to the use of a large amount of acid-base reagents. Summary of the Invention

[0005] In response to the technical problems of existing comprehensive recycling methods for waste yeast, such as large-scale yeast loss and residual acid and alkali reagents, the present invention proposes a comprehensive utilization method for waste beer yeast. The method has the characteristics of simple operation, no need for large amounts of acid and alkali reagents, high nutrient recovery rate and zero waste yeast loss. The method can actually produce three types of yeast β-glucans with different properties and purities and two different types of mannans.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A comprehensive utilization method of waste beer yeast comprises the following steps:

[0008] Primary enzymatic hydrolysis: Weigh an appropriate amount of waste beer yeast to prepare a yeast solution, add complex enzyme A for primary enzymatic hydrolysis, and centrifuge to obtain the supernatant and precipitate;

[0009] Secondary enzymatic hydrolysis: after redissolving the precipitate obtained from the primary enzymatic hydrolysis, adding complex enzyme B for secondary enzymatic hydrolysis, and centrifuging to obtain a supernatant and a precipitate;

[0010] Three-step enzymatic hydrolysis: redissolving the precipitate obtained from the second enzymatic hydrolysis, adding the complex enzyme B to perform three-step enzymatic hydrolysis, and centrifuging to obtain a supernatant and a precipitate;

[0011] Boiling water bath shaking: the precipitates obtained from the three enzymatic hydrolysis steps are redissolved and then subjected to boiling water bath shaking extraction, followed by centrifugation to obtain yeast β-glucan precipitate and yeast polysaccharide-containing supernatant;

[0012] Yeast β-glucan extraction: The yeast β-glucan precipitate is sequentially extracted with 3% NaOH in a boiling water bath and enzymatically hydrolyzed four times, and the supernatant obtained by centrifugation is freeze-dried to obtain water-soluble yeast β-glucan;

[0013] Preparation of yeast polysaccharide: the supernatant containing yeast polysaccharide is freeze-dried to obtain yeast polysaccharide additive or mixed with anhydrous ethanol of the supernatant to obtain yeast mannan protein through precipitation.

[0014] In one embodiment, the primary enzymatic hydrolysis comprises the following steps:

[0015] Waste brewer yeast and clean water are mixed at a material-water ratio of 1:(10-20) to prepare a yeast solution, and complex enzyme A is added to the yeast solution until the total enzyme activity reaches (40,000-60,000) U / g (raw material dry weight). After enzymatic hydrolysis at 50-60°C for 5-7 hours, the supernatant and precipitate are obtained by centrifugation.

[0016] In one embodiment, the complex enzyme A consists of neutral protease and papain, and the addition ratio of the neutral protease and papain is (0.53-1): (0.48-1).

[0017] In one embodiment, the secondary enzymatic hydrolysis comprises the following steps:

[0018] The precipitate obtained by the enzymatic hydrolysis was mixed with clean water at a material-water ratio of 1:(10-40) for re-dissolution, and complex enzyme B was added until the total enzyme activity reached (40000-60000) U / g (precipitate dry weight). After enzymatic hydrolysis at 50-60°C for 7-11 hours, the supernatant and precipitate were obtained by centrifugation.

[0019] The three-step enzymolysis comprises the following steps:

[0020] The precipitate obtained by the secondary enzymatic hydrolysis was mixed with clean water at a material-water ratio of 1:(10-40) for redissolution, and complex enzyme B was added until the total enzyme activity reached (40,000-60,000) U / g (precipitate dry weight). After enzymatic hydrolysis at 50-60°C for 5-7 hours, the supernatant and precipitate were obtained by centrifugation.

[0021] In one embodiment, the complex enzyme B is composed of neutral protease, papain and trypsin, and the addition ratio of the neutral protease, papain and trypsin is (1 / 3-1): (1 / 3-1): (1 / 3-1).

[0022] In one embodiment, the yeast β-glucan in the yeast β-glucan precipitate obtained in the boiling water bath shaking step is yellowish-brown water-insoluble yeast β-glucan, the purity of the yellowish-brown water-insoluble yeast β-glucan is 80±4%, and the extraction rate is 23.0-25.9%.

[0023] In one embodiment, the yeast β-glucan extraction comprises the following steps:

[0024] Boiling water bath extraction: The yeast β-glucan precipitate is extracted in a 3% NaOH boiling water bath for 1 hour, centrifuged, washed with water, centrifuged again, and rinsed with anhydrous ethanol. Finally, the precipitate is centrifuged to obtain the supernatant, and anhydrous ethanol is used for standby use;

[0025] Four enzymatic hydrolysis steps: The precipitate obtained by the final centrifugation in the boiling water bath extraction step is re-dissolved in clean water, β-1,3-glucanase and β-1,6-glucanase are added until the total enzyme activity reaches 10,000 U / g (precipitate dry weight), and the mixture is shaken and extracted at 50-60°C for 10-16 hours. The supernatant is centrifuged and freeze-dried to obtain water-soluble yeast β-glucan.

[0026] In one embodiment, the yeast β-glucan in the precipitate obtained after the boiling water bath extraction step is milky white water-insoluble yeast β-glucan, the purity of the milky white water-insoluble yeast β-glucan is above 90%, and the extraction rate is 14.0-15.9%;

[0027] The water-soluble yeast beta-glucan obtained after the four enzymatic hydrolysis steps is pure white water-soluble yeast beta-glucan with a purity of more than 97% and an extraction rate of 13.0-13.3%.

[0028] In one embodiment, the yeast mannan protein is obtained by mixing the supernatant containing yeast polysaccharide with the supernatant anhydrous ethanol in equal proportions and then precipitating the mixture.

[0029] In one embodiment, the supernatants obtained from the primary enzymatic hydrolysis and secondary enzymatic hydrolysis steps can be directly used to prepare yeast extract after being treated with triple-effect distillation.

[0030] Compared with the prior art, the advantages and positive effects of the present invention are:

[0031] 1. The present invention proposes a method for the comprehensive utilization of spent brewer's yeast. The method uses a combination of neutral, acidic, and alkaline proteases to perform segmented enzymatic hydrolysis on the spent brewer's yeast, resulting in more thorough and time-efficient protein removal from the yeast and more yeast β-glucan remaining in the precipitate. Furthermore, the method provided by the present invention can be used to produce three types of yeast β-glucans with different properties and purities. Furthermore, during the polysaccharide separation stage, a boiling water bath shaking method (non-alkaline separation) is used to simultaneously obtain yeast β-glucan and yeast polysaccharide, avoiding the use of large amounts of acid and alkali reagents. This is not only beneficial to environmental protection, but also prevents acid and alkali reagent residues in the extract, thereby improving its safety.

[0032] 2. The comprehensive utilization method of waste brewer's yeast proposed in the present invention can produce three types of yeast β-glucans with different characteristics and purities: yellow-brown water-insoluble β-glucan (purity of 80% ± 4%, extraction rate of approximately 23.0-25.9%), milky white water-insoluble β-glucan (purity > 90%, extraction rate of approximately 14.0-15.9%), and pure white water-soluble β-glucan (purity > 97%, extraction rate of approximately 13.0-13.3%). Two different types of mannan protein and yeast polysaccharide additives (purity > 60%) can also be produced.

[0033] 3. The comprehensive utilization method of waste beer yeast proposed in the present invention has the characteristics of simple operation, no need for large amounts of acid and alkali reagents, high nutrient recovery rate and zero loss of waste yeast. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of the enzyme screening test provided by the embodiments of the present invention;

[0035] Figure 2 The results of the compound enzyme A ratio test provided in the embodiment of the present invention;

[0036] Figure 3 The results of the screening test for the optimal enzymatic hydrolysis conditions of the complex enzyme A provided in the embodiment of the present invention;

[0037] Figure 4 The results of the compound enzyme B ratio test provided in the embodiment of the present invention;

[0038] Figure 5 The results of the screening test for the optimal enzymatic hydrolysis conditions of the complex enzyme B provided in the embodiment of the present invention;

[0039] Figure 6Schematic diagram of the construction of the composite enzyme system for comprehensive utilization of brewer's yeast provided in an embodiment of the present invention;

[0040] Figure 7 This is an overall process flow chart of the comprehensive utilization method of waste beer yeast provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0041] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0042] The embodiment of the present invention provides a method for comprehensive utilization of waste beer yeast, comprising the following steps:

[0043] S1. Primary enzymatic hydrolysis: Weigh an appropriate amount of waste brewer's yeast to prepare a yeast solution, add complex enzyme A for primary enzymatic hydrolysis, and centrifuge to obtain a supernatant and a precipitate;

[0044] In the above step S1, waste brewer's yeast and clean water are first mixed at a material-to-water ratio of 1:(10-20) to form a yeast solution. Complex enzyme A is then added to the yeast solution until the total enzyme activity reaches (40,000-60,000) U / g (raw material dry weight). After enzymatic hydrolysis at 50-60°C for 5-7 hours, the mixture is centrifuged at 7,000 rpm for 10 minutes. The supernatant is retained for later use. The precipitate is washed once with water and then centrifuged at 7,000 rpm for 10 minutes. The supernatant is combined with the previous supernatant for later use (referred to as "Extract 1"), and the precipitate is set aside for later use.

[0045] Furthermore, the complex enzyme A used in this step is specifically composed of two enzymes, neutral protease and papain, wherein the addition ratio of neutral protease and papain is (0.53-1): (0.48-1). The reason why the present invention uses the above two enzymes as complex enzyme A to perform an enzymatic hydrolysis on the yeast solution is that: ① The yeast cells in the yeast mud after beer brewing contain active proteases and cellulases, and proper utilization can reduce the addition of exogenous enzymes, thereby reducing costs and improving efficiency; however, when performing enzymatic hydrolysis again, it should be noted that the time should not be too long (controlled within 5-7h). If the time is too long, the active cellulase, glucanase, etc. of the yeast mud itself will enzymatically hydrolyze the yeast β-glucan, thereby reducing the extraction rate and yield of yeast β-glucan; ② The higher the enzyme activity, the more enzyme is added. Although this will improve efficiency, considering that the preparation process of the yeast extract itself also requires the strengthening of the Maillard reaction, it is not necessary to complete the enzymatic hydrolysis process of the yeast protein in too short a time (controlling within 5-7h is more reasonable).

[0046] S2, secondary enzymatic hydrolysis: after redissolving the precipitate obtained from the primary enzymatic hydrolysis, adding complex enzyme B for secondary enzymatic hydrolysis, and centrifuging to obtain a supernatant and a precipitate;

[0047] In the above step S2, the precipitate obtained by the primary enzymatic hydrolysis is mixed with clean water at a material-water ratio of 1:(10-40) and then redissolved. Complex enzyme B is added until the total enzyme activity reaches (40,000-60,000) U / g (dry weight of the precipitate). After enzymatic hydrolysis at 50-60°C for 7-11 hours, the supernatant and precipitate are obtained by centrifugation at 7,000 rpm for 10 minutes. The supernatant is reserved (referred to as "extract 2") and the precipitate is reserved for later use.

[0048] Furthermore, the complex enzyme B used in the secondary enzymatic hydrolysis step is composed of three enzymes: neutral protease, papain and trypsin, wherein the addition ratio of neutral protease, papain and trypsin is (1 / 3-1): (1 / 3-1): (1 / 3-1).

[0049] Furthermore, the primary enzymolysis and secondary enzymolysis of the present invention are specifically described as follows:

[0050] First, considering that the main components of yeast cell walls are yeast proteins and various yeast polysaccharides including yeast β-glucan, the first step is to separate the protein and polysaccharides. The purpose of the enzymatic hydrolysis process used in the present invention is to convert the protein into short peptides that are soluble in water, while the yeast β-glucan is insoluble in water for separation.

[0051] Secondly, based on the above mechanism of action, and based on the fact that yeast cells themselves contain endogenous active enzymes, exogenous proteases (i.e., compound enzyme A) are added to hydrolyze yeast cell wall structural proteins, and then the endogenous enzymes are removed by centrifugation (i.e., the primary enzymatic hydrolysis step);

[0052] Finally, after the yeast cell wall structure is enzymatically hydrolyzed, compound enzyme B is added (i.e., exogenous enzymes are used completely) for enzymatic hydrolysis. Compound enzyme B is used for enzymatic hydrolysis twice in total. The purpose is to convert the protein into short peptides and dissolve them in water to achieve sufficient separation of protein and polysaccharide.

[0053] It should also be noted that the higher the total enzyme activity of complex enzyme B, the better. The enzymatic hydrolysis time should also be controlled within a reasonable range. This is because higher enzyme activity means more enzyme is added, which improves efficiency. However, since the yeast extract preparation process itself requires an enhanced Maillard reaction, the enzymatic hydrolysis of yeast proteins does not need to be completed in a very short time. Furthermore, the order of the primary and secondary enzymatic hydrolysis steps in the extraction method of the present invention cannot be reversed. This is because the primary enzymatic hydrolysis utilizes endogenous yeast enzymes and the addition of exogenous proteases to hydrolyze yeast cell wall structural proteins, while the secondary enzymatic hydrolysis is performed entirely with exogenous enzymes, and the centrifugation method removes the endogenous enzymes.

[0054] S3, tertiary enzymatic hydrolysis: redissolving the precipitate obtained from the secondary enzymatic hydrolysis, adding the complex enzyme B to perform tertiary enzymatic hydrolysis, and centrifuging to obtain a supernatant and a precipitate;

[0055] In the above step S3, the precipitate obtained by the secondary enzymatic hydrolysis is mixed with clean water at a material-water ratio of 1:(10-40) for redissolution, and then complex enzyme B is added until the total enzyme activity reaches (40,000-60,000) U / g (precipitate dry weight). After enzymatic hydrolysis at 50-60°C for 5-7 hours, the supernatant and precipitate are obtained by centrifugation.

[0056] Furthermore, the compound enzyme B used in the three enzymatic hydrolysis steps is the same as the compound enzyme B used in the above-mentioned step S2, and is composed of three enzymes: neutral protease, papain, and trypsin, wherein the addition ratio of neutral protease, papain, and trypsin is (1 / 3-1): (1 / 3-1): (1 / 3-1). The present invention uses the same compound enzyme B for two consecutive enzymatic hydrolysis steps for the following reasons: ① In order to improve the purity of the extracted yeast β-glucan, it is necessary to further use protease to remove residual structural proteins; ② Considering that prolonged enzymatic hydrolysis will produce bitter amino acids and bitter short peptides, which is not conducive to the preparation of yeast extract, the enzymatic hydrolysis is terminated within an appropriate time and the supernatant is used to prepare the yeast extract.

[0057] S4, shaking in a boiling water bath: re-dissolving the precipitate obtained by the three enzymatic hydrolysis steps, performing extraction by shaking in a boiling water bath, and centrifuging to obtain a yeast β-glucan precipitate and a supernatant containing yeast polysaccharide (i.e., mannan);

[0058] Analysis of step S4 reveals that it is actually the polysaccharide separation stage. Previously, commonly used methods for isolating yeast β-glucan often used direct NaOH solution for washing, which resulted in a significant loss of mannan. Furthermore, ethanol precipitation from the resulting supernatant by this method failed to precipitate mannan, requiring strong acid neutralization and pH adjustment. Even then, the resulting mannan was yellow-brown (a boiling NaOH bath caramelizes the yeast polysaccharide, discoloring the product). Therefore, direct extraction with NaOH solution is incapable of simultaneously producing both yeast mannan and yeast β-glucan, and the purity of the resulting yeast-β-glucan would not exceed 80%.

[0059] Furthermore, the present invention replaces traditional alkaline separation methods at this stage with boiling water bath shaking extraction, ultimately obtaining a yeast β-glucan precipitate and a yeast polysaccharide-containing supernatant. This method not only reduces the use of acid and alkali reagents, reducing environmental pollution, but also solves the problem of residual acid and alkali reagents. Furthermore, the yeast β-glucan in the yeast β-glucan precipitate in this step is a yellowish-brown, water-insoluble yeast β-glucan with a purity of 80±4% and an extraction yield of 23.0-25.9%.

[0060] Furthermore, the present invention first uses pure water for boiling water bath extraction (as shown in step S4), followed by washing with a NaOH solution (as shown in step S5). This method not only produces more yeast mannan, but also increases the purity of water-insoluble yeast β-glucan and the extraction rate. At the same time, these two steps cannot be reversed: the pure water boiling water bath must be performed first, followed by the NaOH solution boiling water bath. The reason why these two steps cannot be reversed is that if the NaOH boiling water bath is directly performed, the resulting supernatant cannot be precipitated with alcohol to obtain mannan, and the resulting supernatant is yellowish-brown. Even if the pH is adjusted and then precipitated with alcohol, the resulting mannan will also be yellowish-brown, which does not meet product requirements.

[0061] S5. Yeast β-glucan extraction: The yeast β-glucan precipitate is sequentially subjected to 3% NaOH boiling water bath extraction and four enzymatic hydrolysis steps, and the supernatant obtained by centrifugation is freeze-dried to obtain water-soluble yeast β-glucan;

[0062] In the above step S5, the yeast β-glucan extraction specifically includes the following steps:

[0063] (1) Boiling water bath extraction: The yeast β-glucan precipitate was extracted with 3% NaOH in a boiling water bath for 1 h, centrifuged, washed with water, centrifuged again, and rinsed with anhydrous ethanol. Finally, the precipitate was centrifuged and the supernatant was set aside in anhydrous ethanol.

[0064] (2) Quadruple enzymatic hydrolysis: The precipitate obtained by the last centrifugation in the boiling water bath extraction step was redissolved in clean water, and β-1,3-glucanase and β-1,6-glucanase were added to a total enzyme activity of 10,000 U / g (precipitate dry weight). The mixture was extracted by shaking at 50-60°C for 10-16 h, and the supernatant was obtained by centrifugation and freeze-dried to obtain water-soluble yeast β-glucan.

[0065] Based on the above step (1), the yeast β-glucan in the precipitate obtained after the boiling water bath extraction step is milky white water-insoluble yeast β-glucan, the purity of the milky white water-insoluble yeast β-glucan is above 90%, and the extraction rate is 13.0-15.9%.

[0066] Based on the above step (2), it can be seen that this method is actually a stage for improving the water solubility of yeast β-glucan. In this stage, the water solubility of milky white water-insoluble yeast β-glucan is improved by an enzymatic method, and finally yeast β-glucan with better purity and solubility is obtained, that is, pure white water-soluble yeast β-glucan with a purity of more than 97% and an extraction rate of 13.0-13.3%. At the same time, since the main structural types of yeast β-glucan are yeast β-1,3 structure and yeast β-1,6 structure, by selecting specific enzymes according to the structure, yeast β-glucan with high efficiency and high purity can be obtained.

[0067] S6. Preparation of yeast polysaccharide: freeze-dry the supernatant containing yeast polysaccharide to obtain yeast polysaccharide additive or mix it with the supernatant obtained in step S5 in anhydrous ethanol in equal proportions and precipitate it to obtain yeast mannan protein.

[0068] A comprehensive analysis of the boiling water bath extraction steps S5 and S6 shows that ethanol is first used to clean the yeast β-glucan precipitate. The resulting supernatant ethanol solution can then be used to precipitate yeast mannan, significantly reducing extraction costs. It is important to emphasize that this step cannot be reversed. If anhydrous ethanol is used to precipitate yeast mannan first, only a 50% ethanol solution is obtained, which fails to effectively clean and purify the yeast β-glucan. Therefore, the order cannot be reversed.

[0069] It should be further explained that, through analysis of the above-mentioned extraction method, it can be seen that three types of yeast β-glucans with different characteristics and purities are prepared in the boiling water bath shaking step, the 3% NaOH boiling water bath extraction step, and the four enzymatic hydrolysis steps, namely, yellowish-brown water-insoluble β-glucan (purity of 80%±4%, extraction rate of approximately 25.9%), milky-white water-insoluble β-glucan (purity>90%, extraction rate of approximately 15.9%), and pure white water-soluble β-glucan (purity>97%, extraction rate of approximately 13.3%). In the yeast polysaccharide preparation step, two different types of mannan protein and yeast polysaccharide additives can be prepared. Those skilled in the art can prepare related products with different characteristics according to actual needs.

[0070] In one embodiment, the supernatants obtained from the primary and secondary enzymatic hydrolysis steps (i.e., "Extract 1" and "Extract 2") can be directly used to prepare yeast extract after triple-effect distillation. Specifically, 5-phosphodiesterase and adenosine deaminase can be added to Extract 1 and Extract 2 to prepare I+G type yeast extract (the national standard requires I+G ≥ 2%). Plant-derived I+G can also be added.

[0071] In order to more clearly and in detail introduce the comprehensive utilization method of waste beer yeast provided by the embodiment of the present invention, it will be described below with reference to specific embodiments.

[0072] Example 1

[0073] This embodiment provides a method for comprehensive utilization of waste brewer's yeast, specifically a method for preparing a yellow-brown water-insoluble β-glucan and yeast polysaccharide additive, comprising the following steps:

[0074] (1) Primary enzymatic hydrolysis: Weigh 1 g of yeast powder (i.e., waste yeast sludge is calculated as 1 g dry weight) and add 20 ml of water to prepare yeast solution. Add complex enzyme A (neutral protease: papain = 0.5253:0.4747) with a total enzyme activity of 40,000 U. After hydrolysis at 60°C for 5 h, centrifuge at 7,000 rpm for 10 min, transfer the supernatant to extract 1, and wash the precipitate once with 20 ml of water. Centrifuge at 7,000 rpm for 10 min, transfer the supernatant to extract 1, and precipitate for use.

[0075] (2) Secondary enzymatic hydrolysis: The precipitate obtained in step (1) was redissolved in 40 ml of water, and complex enzyme B (neutral protease: papain: trypsin = 1 / 3:1 / 3:1 / 3) was added until the total enzyme activity reached 40,000 U / g (precipitate dry weight). The product was enzymatically hydrolyzed at 60°C for 11 h, and then centrifuged at 7,000 rpm for 10 min. The supernatant was transferred to extract 2, and the precipitate was set aside for use.

[0076] (3) Three-step enzymatic hydrolysis: The precipitate obtained in step (2) was redissolved in 40 ml of water, and complex enzyme B (neutral protease: papain: trypsin = 1 / 3:1 / 3:1 / 3) was added until the total enzyme activity reached 40,000 U / g (precipitate dry weight). After enzymatic hydrolysis at 60°C for 7 h, the precipitate was centrifuged at 7,000 rpm for 10 min and the precipitate was set aside for use;

[0077] (4) Preparation of yellowish-brown water-insoluble β-glucan: The precipitate obtained in step (3) was re-mixed with a water-to-solid ratio of 40:1, subjected to 5-h boiling water bath shaking extraction, and centrifuged at 7000 rpm for 10 min to obtain a yeast β-glucan precipitate and a supernatant containing yeast polysaccharide. At this time, the yeast β-glucan in the yeast β-glucan precipitate was yellowish-brown water-insoluble β-glucan with a purity of 80% ± 4% and an extraction rate of approximately 25.9%.

[0078] (5) Preparation of milky white water-insoluble β-glucan: The yeast β-glucan precipitate obtained in step (4) was extracted with 3% NaOH in a boiling water bath for 1 hour, and then centrifuged at 7000 rpm for 10 minutes to obtain a precipitate, which was rinsed once with clean water and then centrifuged at 7000 rpm for 10 minutes; rinsed once with anhydrous ethanol and then centrifuged at 7000 rpm for 10 minutes to obtain a precipitate, and the supernatant was anhydrous ethanol for standby use; at this time, the purity of the yeast β-glucan in the precipitate was milky white water-insoluble β-glucan, the purity of which was >90%, and the extraction rate was about 15.9%;

[0079] (6) Preparation of pure white water-soluble β-glucan: The precipitate was re-dissolved in 40 ml of water, and β-1,3-glucanase and β-1,6-glucanase were added until the total enzyme activity reached 10,000 U / g (precipitate dry weight). The mixture was extracted by shaking at 60°C for 16 h, and centrifuged at 7,000 rpm for 10 min to obtain the supernatant. The supernatant was freeze-dried to prepare water-soluble yeast β-glucan. At this time, the yeast β-glucan was pure white water-soluble β-glucan with a purity of >97% and an extraction rate of approximately 13.3%.

[0080] (7) Preparation of yeast polysaccharide additive: The supernatant containing yeast polysaccharide obtained in step (4) is directly freeze-dried to obtain the yeast polysaccharide additive;

[0081] (8) Preparation of mannan protein: The supernatant containing yeast polysaccharide obtained in step (4) was mixed with the supernatant obtained in step (5) with anhydrous ethanol in a ratio of 1:1, and the yeast mannan protein was precipitated;

[0082] (9) Yeast extract can be prepared by triple-effect distillation of the extract 1 and extract 2 obtained in steps (1) and (2).

[0083] Complex enzyme A and complex enzyme B screening test

[0084] 1. Enzyme type screening test

[0085] In order to obtain the optimal components of complex enzyme A and complex enzyme B, this screening test selected enzymes with broad specificity from neutral protease, alkaline protease, and acidic protease, and conducted factorial experimental screening. The enzyme selection was based on high activity and low addition amount to ensure that the added enzymes caused as little interference with the extraction purity as possible. The specific test is as follows:

[0086] 1.1 Types of enzymes used

[0087] Table 1 Characteristics of different enzymes

[0088] name Restriction site Product enzyme activity Neutral protease Broad specificity 1000 U / mg Papain Broad specificity during long-term incubation 2000 U / mg Trypsin After lysine and arginine 2500 U / mg Pepsin Broad specificity 3500 U / mg

[0089] 1.2 Test methods

[0090] The amount of yeast powder was fixed at 1 g, the total enzyme activity was 10,000 U, a neutral solution was added, and after heating at 60°C for 1 hour, the optimal enzyme was screened by counting three times based on the average sugar content.

[0091] 1.3 Enzyme type screening test results

[0092] In this experiment, papain and pepsin were selected as acidic proteases, and trypsin and carbsberg protease were selected as alkaline proteases. The total sugar content in the precipitate after the action time was measured to compare the comprehensive hydrolysis activity of proteases (the results are shown in Figure 2). Figure 1 As shown), neutral protease, trypsin (alkaline protease), and papain (acidic protease) were finally determined as alternative components.

[0093] 2. Compound Protease A Ratio Experiment:

[0094] 2.1 Six Sigma theory design mixture experiment:

[0095] According to the Six Sigma theory, a mixture experiment was designed with simple centroids and a total of 10 groups, as shown in the table below:

[0096] Table 2 Six Sigma Theory Experimental Design

[0097]

[0098]

[0099] 2.2 Test method:

[0100] The fixed substrate was 1 g, the total enzyme activity was added to 40,000 U, and after heating at 60°C for 6 h, the optimal enzyme was screened by counting three times and using the average precipitated sugar concentration (Y).

[0101] 2.3 Test results:

[0102] After analyzing the relevant data using Minitab software, the optimal ratio of composite protease was determined to be: neutral protease: papain = 0.5253:0.4747. The test results are as follows Figure 2 shown.

[0103] 3. Screening test of optimal enzymatic hydrolysis conditions of complex enzyme A

[0104] 3.1 Test Purpose:

[0105] Six Sigma theory was used to design a five-factor two-level study to investigate the effects of water-to-material ratio, enzymatic hydrolysis time (h), number of centrifugal extractions, temperature (℃), and total enzyme activity (U / g) on ​​polysaccharide extraction rate.

[0106] 3.2 Experimental Grouping:

[0107] Minitab was used to design a 5-factor 2-level study with 1 center point and 17 groups in total. Y was the total sugar concentration in the precipitate.

[0108] Table 3 Six Sigma five-factor two-level experiment

[0109]

[0110]

[0111] 3.3 Test method:

[0112] The solution pH was fixed without acid-base adjustment, and the optimal conditions were evaluated based on the glucan extraction rate.

[0113] 3.4 Test results:

[0114] By studying the effects of water-to-material ratio, enzymatic hydrolysis time, number of centrifugal extractions, temperature, and total enzyme activity on the polysaccharide residue after cell wall destruction, it was found that the interactions among various factors were significant. The optimal enzymatic hydrolysis conditions were finally determined to be: water-to-material ratio: 20:1, enzymatic hydrolysis at 60°C for 5h, 1 extraction, and enzyme addition until the total enzyme activity reached 40,000U / g (raw material dry weight). The test results are shown in Figure 3 In addition, this experiment found that the enzyme activity and enzyme addition should not be too high, which will affect the cost and the purity of polysaccharide extraction; the water-to-material ratio should not be too low, which will prevent complete suspension; and the time should not be too low, which will lead to excessive protein residues and affect the subsequent purification and extraction of polysaccharides.

[0115] 4. Compound enzyme B ratio test

[0116] 4.1 Test Groups:

[0117] According to the Six Sigma theory, a mixture experiment was designed with simple centroids and a total of 10 groups, as shown in the table below:

[0118] Table 4 Six Sigma Mixture Experimental Design

[0119] Serial number Standard sequence Run sequence Point Type Block Neutral protease Papain Trypsin 1 1 1 1 1 1.00000 0.00000 0.00000 2 2 2 1 1 0.00000 1.00000 0.00000 3 3 3 1 1 0.00000 0.00000 1.00000 4 4 4 2 1 0.50000 0.50000 0.00000 5 5 5 2 1 0.50000 0.00000 0.50000 6 6 6 2 1 0.00000 0.50000 0.50000 7 7 7 0 1 0.33333 0.33333 0.33333 8 8 8 -1 1 0.66667 0.16667 0.16667 9 9 9 -1 1 0.16667 0.66667 0.16667 10 10 10 -1 1 0.16667 0.16667 0.66667

[0120] 4.2 Test method:

[0121] The fixed substrate (and the dry weight of the precipitate after enzymatic hydrolysis by complex enzyme A) was 1 g, the total enzyme activity was added to 40,000 U, and after heating at 60°C for 6 h, the optimal enzyme was screened by counting three times and using the average precipitated sugar concentration (Y).

[0122] 4.3 Test results:

[0123] After analysis with Minitab software, the optimal ratio of compound protease was determined as follows: neutral protease: papain: trypsin = 1 / 3: 1 / 3: 1 / 3. The test results are as follows: Figure 4 shown.

[0124] 5. Screening test of optimal enzymatic hydrolysis conditions of complex enzyme B

[0125] 5.1 Test method:

[0126] The optimal enzymatic hydrolysis time and optimal enzymatic hydrolysis temperature were selected by single-factor experiments respectively. The optimal enzymatic hydrolysis temperature was selected by fixing the enzymatic hydrolysis for 6 h, and the optimal enzymatic hydrolysis time was selected by fixing the enzymatic hydrolysis temperature at 60°C.

[0127] 5.2 Test results:

[0128] The optimal enzymatic hydrolysis temperature was determined to be 60°C and the optimal enzymatic hydrolysis time was 18h. The test results are as follows: Figure 5 shown.

[0129] In summary, two enzyme systems including complex enzyme A and complex enzyme B were constructed through the above five experiments, wherein the total enzymatic hydrolysis time was 23h (complex enzyme A: 5h + complex enzyme B: 18h), and the ratio of complex enzyme A was: neutral protease: papain = 0.5253:0.4747; the ratio of complex enzyme B was: neutral protease: papain: trypsin = 1 / 3:1 / 3:1 / 3. However, in order to realize the comprehensive utilization of brewer's yeast, the present invention needs to collect and concentrate the yeast extract. If only the supernatant after the final protein is taken out is taken, it will cause excessive hydrolysis of the protein, and more bitter substances (valine, leucine, isoleucine, phenylalanine, histidine, arginine, methionine) will be released, which is not conducive to the production and preparation of yeast extract. Therefore, it is chosen to terminate the enzymatic hydrolysis once when the total enzymatic hydrolysis time is 16h, and collect the supernatant for the production and preparation of yeast extract (the complex enzyme system is constructed as shown in FIG). Figure 6 shown).

Claims

1. A comprehensive utilization method of waste beer yeast, characterized in that: The following steps are involved: Primary enzymatic hydrolysis: Weigh an appropriate amount of waste beer yeast to prepare a yeast solution, add complex enzyme A for primary enzymatic hydrolysis, and centrifuge to obtain the supernatant and precipitate; Secondary enzymatic hydrolysis: after redissolving the precipitate obtained from the primary enzymatic hydrolysis, adding complex enzyme B for secondary enzymatic hydrolysis, and centrifuging to obtain a supernatant and a precipitate; Three-step enzymatic hydrolysis: redissolving the precipitate obtained from the second enzymatic hydrolysis, adding the complex enzyme B to perform three-step enzymatic hydrolysis, and centrifuging to obtain a supernatant and a precipitate; Boiling water bath shaking: the precipitates obtained from the three enzymatic hydrolysis steps are redissolved and then subjected to boiling water bath shaking extraction, followed by centrifugation to obtain yeast β-glucan precipitate and yeast polysaccharide-containing supernatant; Yeast β-glucan extraction includes the following steps: Boiling water bath extraction: The yeast β-glucan precipitate is extracted in a boiling water bath with 3% NaOH for 1 hour, centrifuged, washed with water, centrifuged again, and rinsed with anhydrous ethanol. Finally, the precipitate is centrifuged to obtain a precipitate, and the supernatant is prepared with anhydrous ethanol for later use. Four enzymatic hydrolysis steps: The precipitate obtained by the final centrifugation in the boiling water bath extraction step is re-dissolved in clean water, β-1,3-glucanase and β-1,6-glucanase are added, and enzymatic hydrolysis is performed. The supernatant obtained by centrifugation is then freeze-dried to obtain water-soluble yeast β-glucan. Preparation of yeast polysaccharide: freeze-drying the supernatant containing yeast polysaccharide to obtain yeast polysaccharide additive or mixing the supernatant with anhydrous ethanol and precipitating to obtain yeast mannan protein; The complex enzyme A is composed of neutral protease and papain; The complex enzyme B consists of neutral protease, papain and trypsin.

2. The comprehensive utilization method of waste beer yeast according to claim 1, characterized in that: The primary enzymatic hydrolysis comprises the following steps: Waste brewer's yeast and clean water are mixed at a material-water ratio of 1:(10-20) to prepare a yeast solution, and complex enzyme A is added to the yeast solution until the total enzyme activity reaches (40,000-60,000) U / g dry weight of the raw material. After enzymatic hydrolysis at 50-60°C for 5-7 hours, the supernatant and precipitate are obtained by centrifugation.

3. The comprehensive utilization method of waste beer yeast according to claim 2, characterized in that: In the complex enzyme A, the addition ratio of the neutral protease and papain is (0.53-1): (0.48-1).

4. The comprehensive utilization method of waste beer yeast according to claim 1, characterized in that: The secondary enzymolysis comprises the following steps: The precipitate obtained by the primary enzymatic hydrolysis was mixed with clean water at a material-water ratio of 1:(10-40) and then re-dissolved, and complex enzyme B was added until the total enzyme activity reached (40,000-60,000) U / g dry weight of the precipitate. After enzymatic hydrolysis at 50-60°C for 7-11 hours, the supernatant and precipitate were obtained by centrifugation. The three-step enzymolysis comprises the following steps: The precipitate obtained from the secondary enzymatic hydrolysis was mixed with clean water at a material-water ratio of 1:(10-40) and re-dissolved, and complex enzyme B was added until the total enzyme activity reached (40,000-60,000) U / g dry weight of the precipitate. After enzymatic hydrolysis at 50-60°C for 5-7 hours, the supernatant and precipitate were obtained by centrifugation.

5. The comprehensive utilization method of waste beer yeast according to claim 4, characterized in that: The addition ratio of the neutral protease, papain and trypsin is (1 / 3-1): (1 / 3-1): (1 / 3-1).

6. The comprehensive utilization method of waste beer yeast according to claim 1, characterized in that: The yeast β-glucan in the yeast β-glucan precipitate obtained in the boiling water bath shaking step is yellowish-brown water-insoluble yeast β-glucan, the purity of the yellowish-brown water-insoluble yeast β-glucan is 80%±4%, and the extraction rate is 23.0%-25.9%.

7. The comprehensive utilization method of waste beer yeast according to claim 1, characterized in that: During the four enzymatic hydrolysis steps, β-1,3-glucanase and β-1,6-glucanase were added until the total enzyme activity reached 10,000 U / g of precipitate dry weight. The mixture was shaken and extracted at 50-60° C. for 10-16 hours. The supernatant was centrifuged and freeze-dried to obtain water-soluble yeast β-glucan.

8. The comprehensive utilization method of waste beer yeast according to claim 1, characterized in that: The yeast β-glucan in the precipitate obtained after the boiling water bath extraction step is milky white water-insoluble yeast β-glucan, the purity of the milky white water-insoluble yeast β-glucan is above 90%, and the extraction rate is 14.0%-15.9%; The water-soluble yeast β-glucan obtained after the four enzymatic hydrolysis steps is pure white water-soluble yeast β-glucan with a purity of more than 97% and an extraction rate of 13.0%-13.3%.

9. The comprehensive utilization method of waste beer yeast according to claim 1, characterized in that: The yeast mannan protein is obtained by mixing the supernatant containing yeast polysaccharide with the supernatant anhydrous ethanol in equal proportions and precipitating the mixture.

10. The comprehensive utilization method of waste beer yeast according to claim 1, characterized in that: The supernatants obtained from the primary enzymolysis and secondary enzymolysis steps are directly used to prepare yeast extract after being treated with triple-effect distillation.

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