A method for preparing yeast protein using low-concentration biomass saccharification liquid

Abstract

The present invention relates to the field of yeast fermentation technology, and specifically to a method for preparing yeast protein using low-concentration biomass saccharification liquid. The present invention provides a method for preparing yeast protein using low-concentration biomass saccharification liquid, comprising the following steps: S1, steam explosion treatment and saccharification treatment of biomass to obtain biomass saccharification liquid; S2, biomass saccharification liquid and nutrients are mixed into a mixed solution, wherein the nutrients include trace elements and vitamins; S3, yeast strains are fermented using the mixed solution obtained in step S2 as a carbon source to obtain yeast protein. The method of the present invention has a stable fermentation process, high production efficiency, and stable product quality, and realizes the industrial application of low-concentration biomass saccharification liquid to produce yeast, alleviating the problem of molasses resource shortage in the yeast industry.

Description

Technical Field

[0001] The invention relates to the technical field of yeast fermentation, and in particular to a method for preparing yeast protein by utilizing low-concentration biomass saccharification liquid. Background Art

[0002] Biomass is the most abundant renewable resource on earth. The annual photosynthesis product in the world is as high as 150-200 billion tons, of which more than 80% are lignocellulosic substances (such as various grasses, trees, etc. are all complexes of cellulose, hemicellulose and lignin). From the chemical composition point of view, the main components of these lignocellulosic materials are sugars and aromatic compounds. In nature, microorganisms will degrade them into energy and other biomass, and finally decompose them into carbon dioxide and water. Generally speaking, the conversion and utilization technology of biomass resources can be roughly divided into three categories: one is direct combustion, the second is biological conversion technology, and the third is thermochemical technology to convert into high-quality fluid fuels. Among them, the biological conversion of biomass resources is a hot topic of research. The biological conversion and utilization of biomass resources must go through three basic steps: decomposition of biomass resources, microbial fermentation, and separation and purification of metabolites.

[0003] Chinese patent CN102351817B discloses a method for biochemical furfural and ethanol from plant fiber materials, comprising: catalytically hydrolyzing the fiber raw material, filtering and separating, and removing enzymes to obtain pentosan hydrolyzate; using a binary composite solid acid catalyst to dehydrate and cyclize the obtained hydrolyzate to obtain a furfural product; taking the solid residue obtained in the above steps, adding a buffer solution of pH 4 to 6 at a liquid-solid ratio of 9:1 to 5:1, sterilizing, adding cellulase at an enzyme dosage of 40 to 100 IU / g substrate, enzymolysis for 24 to 48 hours, filtering and separating the hydrolysis residue, and removing enzyme proteins from the filtrate to obtain a glucose solution of the cellulose hydrolyzate. Liquid yeast cell culture medium is added to the cellulose hydrolyzate obtained in the above steps, sterilized, inoculated with 0.5 to 1.5% saccharomyces cerevisiae seed culture medium, fermented and cultured for 36 to 48 hours, centrifuged to recover the yeast cells, and the supernatant is the ethanol product.

[0004] Chinese patent CN101173302A discloses an inert carrier material for solid-state fermentation and a preparation method thereof. The inert carrier material is a porous solid material synthesized from biomass polyol, isocyanate, triethylenediamine, dibutyltin bicarbonate, Tween 80 and water. The preparation method is as follows: steam-exploded plant straw material is added with polyhydroxy alcohol and sulfuric acid, and liquefied for 1 to 2 hours under stirring conditions at a temperature of 110 to 150° C. to obtain a straw liquefied product; the liquefied product is then dissolved with industrial alcohol, the residue is filtered off, and the biomass polyol is concentrated; the biomass polyol is then added with isocyanate, triethylenediamine, dibutyltin bicarbonate, Tween 80 and water to generate an inert carrier material for solid-state fermentation; the plastic has good mechanical properties and high hydrophilicity; the method has relatively mild conditions, low equipment investment, and can utilize agricultural waste, with high conversion efficiency and low price.

[0005] Chinese patent CN104593448B discloses a method for producing ethanol from lignocellulosic biomass, the steps of which are as follows: (1) crushing the raw material, adding sulfuric acid solution for normal pressure heat treatment, centrifuging or filtering to separate the acid hydrolyzate and the residue; (2) placing the residue in water, adding calcium hydroxide or calcium oxide, and normal pressure heat treatment to obtain a mixture A; (3) adding the acid hydrolyzate to the mixture A to obtain a mixture B, heat treatment at 50°C to 100°C for 0.5 to 2h, cooling to 25-45°C, and adjusting the pH to 3.5 to 5.5; (4) adding cellulase to the mixture B, and adding nitrogen source and nutrient salt at the same time, inoculating 2 to 8% of activated xylose fermentation bacteria and 1 to 4% of brewer's yeast according to the volume of the mixture B, and performing simultaneous saccharification and fermentation to produce ethanol. The present invention effectively reduces the content of fermentation inhibitors generated in the pretreatment process and improves the conversion yield of ethanol by coordinating the pretreatment, enzymolysis, fermentation and other steps in the process of converting biomass into ethanol.

[0006] By consulting patents and literature reports, we found that the current technologies related to biomass resource utilization are mainly related to bio-power generation, fuel ethanol and other related production. The fields related to yeast production are also mainly used for alcohol fermentation and screening of related strains such as pentose sugars. The fields related to protein production are also mainly based on biomass as raw materials, using microbial strains for solid-state fermentation to produce protein raw materials. If biomass resources can be used to prepare yeast protein, it can effectively alleviate the problem of molasses resource shortage in the yeast industry, and can also alleviate the problem of protein resource shortage and "human-animal competition for food" in the feed industry. At the same time, the utilization of biomass resources can reduce environmental pollution, which is in line with the development requirements of the country's "dual carbon strategy". Summary of the invention

[0007] The technical problem to be solved by the present invention is as follows: The present invention provides a method for preparing yeast protein by using low-concentration biomass saccharification liquid, which has high production efficiency and stable product quality.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing yeast protein using low-concentration biomass saccharification liquid, comprising the following steps:

[0010] S1, steam explosion and saccharification of biomass to obtain biomass saccharification liquid;

[0011] S2, preparing a mixed solution of biomass saccharification liquid and nutrients, wherein the nutrients include trace elements and vitamins;

[0012] S3, fermenting the yeast strain using the mixed solution obtained in step S2 as a carbon source to obtain yeast protein.

[0013] Preferably, in the above method, the trace element is one or more of copper, zinc, iron, magnesium, potassium or calcium; preferably, the vitamin is any one or more of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6 and biotin.

[0014] Preferably, the biomass in step S1 is lignocellulosic biomass; preferably, the lignocellulosic biomass is selected from one or more of bagasse, straw, rice straw, corn cob, cotton stalk, wood chips and wheat straw.

[0015] Preferably, in the above method, the reducing sugar concentration in the biomass saccharification liquid in step S1 is 2-9wt%.

[0016] Preferably, in the above method, based on the mass of the mixed solution, the nutrients include 3-5 ppm copper, 50-100 ppm zinc, 50-100 ppm iron, 2000-10000 ppm potassium, 2000-10000 ppm calcium, 2000-10000 ppm magnesium, 0-50 ppm vitamin B1, 1-3 ppm vitamin B2, 10-50 ppm vitamin B3, 50-100 ppm vitamin B5, 10-50 ppm vitamin B6 and 10-100 ppm biotin.

[0017] Preferably, in the above method, the steam explosion is continuous steam explosion or instantaneous ejection steam explosion; preferably, the pressure of the continuous steam explosion is 1.0-1.6 MPa, and the cooking time is more preferably 5-10 min; or preferably, the pressure of the instantaneous ejection steam explosion is 1.0-1.4 MPa, and the cooking time is more preferably 2-6 min.

[0018] Preferably, in the above method, the saccharification treatment uses hemicellulase and / or cellulase for enzymatic hydrolysis; preferably, the enzyme is added in an amount of 1-4% based on the dry weight of the material after steam explosion, and the enzyme activity is preferably ≥10000 U / g.

[0019] Further preferably, in the above method, the enzymatic hydrolysis pH is 5.0-6.0, and the enzymatic hydrolysis temperature is 40-60° C.; more preferably, the enzymatic hydrolysis time is 24-72 h.

[0020] Preferably, in the above method, the saccharification treatment further includes a step of separating the biomass saccharification liquid.

[0021] Preferably, the yeast species is selected from one or more of Saccharomyces cerevisiae, Kluyveromyces marxii and Candida utilis, preferably Candida utilis.

[0022] Preferably, in the above method, the fermentation temperature is 28-32°C; preferably, the fermentation pH is 4.5-5.5, and more preferably, the fermentation time is 72-120h.

[0023] Preferably, in the above method, the fermentation nitrogen source is selected from one or more of urea, ammonium sulfate, ammonia water or yeast extract.

[0024] Preferably, in the above method, the fermentation phosphorus source is selected from one or more of ammonium phosphate, monoammonium hydrogen phosphate, diammonium hydrogen phosphate and phosphoric acid.

[0025] Preferably, in the above method, the fermentation is continuous fermentation.

[0026] Preferably, in the above method, the flow acceleration of the mixed solution in step S2 is 4 to 10 kg / h; preferably, the flow acceleration of the nitrogen source is 0.5 to 0.8 kg / h, and the flow acceleration of the phosphorus source is 0.5 to 0.8 kg / h;

[0027] Further preferably, the specific growth rate of the yeast strain during the fermentation process is 0.2 to 0.4 h ～1 .

[0028] The present invention also provides a yeast protein prepared by the above method, wherein the protein content is 50-60wt%, and the RNA content is 5-12wt%; preferably, the protein content is 52-58wt%, and the RNA content is 8-12wt%.

[0029] Beneficial effects of the present invention:

[0030] The method of the present invention is used to produce yeast protein, and the fermentation process is stable, the production efficiency is high, and the quality of the produced product is stable; compared with the prior art method of producing yeast using molasses, the method of the present invention is equivalent to the prior art in terms of production efficiency and quality. Therefore, the method of the present invention can realize the industrial application of preparing yeast protein from low-concentration biomass saccharification liquid, increase the channels for obtaining yeast protein sugar resources, broaden the application scope of low-concentration biomass saccharification liquid, and at the same time alleviate the problem of molasses resource shortage in the yeast industry, and also increase the application scope of biomass saccharification liquid. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and technical effect of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention is described clearly and completely. The embodiments described below are part of the embodiments of the present invention, not all of them. In combination with the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The present invention uses steam explosion technology for pretreatment, and enzymatically saccharifies the biomass pretreatment liquid by hemicellulase and / or cellulase to obtain biomass saccharification liquid. Compared with the fermentation carbon sources such as molasses, starch hydrolyzed sugar, etc. currently used in the yeast protein industry, the reducing sugar concentration of the biomass saccharification liquid is low, and the relevant nutrient elements are lacking, which is not conducive to the reproduction and metabolism of yeast. By adding nutrient elements with specific composition and content, optimizing the culture medium components and culture conditions, finally achieving efficient production of yeast protein under low sugar concentration conditions.

[0033] In a specific embodiment of the present invention, the present invention provides a method for preparing yeast protein using low-concentration biomass saccharification liquid, comprising the following steps:

[0034] S1, steam explosion and saccharification of biomass to obtain biomass saccharification liquid;

[0035] S2, preparing a mixed solution of biomass saccharification liquid and nutrients, wherein the nutrients include trace elements and vitamins;

[0036] S3, fermenting the yeast strain using the mixed solution obtained in step S2 as a carbon source to obtain yeast protein.

[0037] Wherein, the reducing sugar concentration of the biomass saccharification liquid in step S1 is 2-9wt%; in step S2, the trace elements are one or more of copper, zinc, iron, magnesium, potassium or calcium; the vitamins are any one or more of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6 and biotin. Based on the mass of the mixed solution, the nutrients include 3-5ppm copper, 50-100ppm zinc, 50-100ppm iron, 2000-10000ppm potassium, 2000-10000ppm calcium, 2000-10000ppm magnesium, 0-50ppm vitamin B1, 1-3ppm vitamin B2, 10-50ppm vitamin B3, 50-100ppm vitamin B5, 10-50ppm vitamin B6 and 10-100ppm biotin.

[0038] According to the present invention, the biomass is any biomass raw material containing lignocellulose, for example including but not limited to one or more of bagasse, straw, rice stalks, corn cobs, cotton stalks, wood chips and wheat straw.

[0039] The steam explosion treatment method of the present invention is not particularly limited, as long as the biomass can be released to normal pressure. For example, the biomass raw material can be sprayed out through a spray valve to reduce the pressure to normal pressure. The steam explosion can be continuous steam explosion or instantaneous ejection steam explosion.

[0040] According to the present invention, the saccharification treatment further includes a step of separating the biomass saccharification liquid to remove insoluble matter in the sugar liquid, and further modulating to obtain sugar liquids of different concentrations, which can further improve the fermentation efficiency and product quality.

[0041] According to the present invention, in order to improve the fermentation effect, a nitrogen source and / or a phosphorus source are further added during the fermentation process, wherein the nitrogen source is selected from one or more of urea, ammonium sulfate, ammonia water or yeast extract; and the phosphorus source is selected from one or more of ammonium phosphate, monoammonium hydrogen phosphate, diammonium phosphate and phosphoric acid.

[0042] According to the present invention, in order to further efficiently produce yeast protein, the high specific production rate characteristics of different yeast strains are fully utilized, and continuous fermentation technology is used to produce yeast protein under low sugar concentration.

[0043] Preferably, the flow acceleration of the mixed solution is 4-10 kg / h; the flow acceleration of the nitrogen source is 0.5-0.8 kg / h, and the flow acceleration of the phosphorus source is 0.5-0.8 kg / h; further preferably, the fermentation process specific growth rate is 0.2-0.4 h -1 .

[0044] The source of yeast used in the present invention is not particularly limited, including but not limited to Saccharomyces cerevisiae, Kluyveromyces marx and Candida utilis yeast, etc., preferably Candida ruan. Candida ruan has a high specific growth rate characteristic, which can improve the fermentation efficiency under low sugar concentration conditions. The Candida ruan (CICC 31170) used in the embodiment of the present invention was purchased from the China Industrial Microbiological Culture Collection Management Center (CICC) (online purchase query address: http: / / m.china-cicc.org / ).

[0045] In a preferred embodiment of the present invention, step s3 further comprises the step of multi-stage culturing of the yeast strain, which specifically comprises the following steps:

[0046] (1) Slant culture: The preserved yeast strains were inoculated into a sucrose culture medium, the pH was adjusted to 4.5-5.0, and the culture was carried out at a temperature of 28-32° C. for 12-36 h; the composition of the culture medium was 10 wt% sucrose, 2 wt% yeast extract, 1 wt% magnesium sulfate, 1 wt% diammonium phosphate, and the remainder was water;

[0047] (2) Primary seed culture (liquid seed culture): The yeast strain cultured on the slant is inoculated into a special culture bottle containing 1000 mL, inoculated into a sucrose medium, the pH is adjusted to 4.5-5.0, and the culture time is 12-24 hours at a temperature of 28-32°C; the composition of the medium is sucrose 10wt%, yeast extract 2wt%, magnesium sulfate 1wt%, ammonium dihydrogen phosphate 1wt%, and the rest is water;

[0048] (3) Secondary seed culture (fermentation tank seed culture): 2 L of liquid seed culture was inoculated into a 45 L fermentation tank filled with 18 L of bottom water, the pH was adjusted to 4.5-5.0, and fermentation was carried out at 28-32°C for 12-36 h, wherein the carbon source, nitrogen source, phosphorus source and pH regulator were all added by flow addition, the carbon source was 30% molasses, the flow acceleration was 0.125-0.2 L / h, the nitrogen source was ammonium sulfate, the flow acceleration was 8-20 g / h, and the phosphorus source was ammonium phosphate, the flow acceleration was 2-4 g / h. After the fermentation was completed, the yeast strain was obtained by centrifugation.

[0049] It should be noted that the culture medium for the slant culture, primary seed culture and secondary seed culture stages of the present invention is not particularly limited, and any culture medium known in the art as long as it is suitable for the growth of yeast strains can be used.

[0050] The present invention also provides a yeast protein prepared by the method, wherein the protein content of the yeast protein is 50-60wt% and the RNA content of the yeast protein is 5-12wt%.

[0051] The beneficial effects of the method for preparing yeast protein using low-concentration biomass saccharification liquid of the present invention are further illustrated by specific examples below.

[0052] The sources of raw materials and equipment used in the examples and comparative examples of the present invention are shown in Table 1.

[0053] Table 1 Raw materials used in the examples of the present invention and the comparative examples

[0054]

[0055] The protein and RNA contents in the yeast and the reducing sugar concentration in the biomass saccharification liquid or molasses in the Examples and Comparative Examples were determined by the following methods:

[0056] 1) Determination of protein content in yeast

[0057] Using the Kjeldahl nitrogen determination method 6.4 in the national standard GB / T 23530-2009, take a sample (equivalent to 30-440 mg of total nitrogen), add 20 mL of concentrated sulfuric acid to digest it in the presence of 5 g of mixed catalyst a (potassium sulfate and copper sulfate pentahydrate mixed in a ratio of 97:3) and 2.5 g of catalyst b (selenium powder and potassium sulfate mixed in a ratio of 0.1:100); then distill it and absorb the product ammonia with boric acid; then titrate it with 0.1 mol / L hydrochloric acid, read the data, and calculate the total nitrogen content.

[0058] Protein content = total nitrogen * 6.25.

[0059] 2) Determination of RNA content in yeast

[0060] 2.1 Reagents

[0061] 0.5 mol / l perchloric acid (HClO4) solution: Add 21.5 mL 70% (or 22.1 mL 68%) perchloric acid to 400 mL distilled water, and then dilute to 500 mL with distilled water.

[0062] 0.25mol / l perchloric acid (HClO4) solution: Take 250ml 0.5mol / l perchloric acid (HClO4) and dilute it to 500ml with distilled water.

[0063] 2.2 Equipment:

[0064] Electronic balance: accurate to 1mg; centrifuge: 4000 rpm, capable of separating yeast; centrifuge tubes, capacity at least 10ml; spectrophotometer: wavelength adjustable to 260nm; hot water bath: 70℃; cold water bath: 4℃; 10ml and 1ml pipettes; 100ml volumetric flask.

[0065] 2.3 Operation steps

[0066] 2.3.1 Sample processing

[0067] Weigh 0.06-0.15g of dry yeast (0.4-0.8g of 18% yeast milk; 1.5-3.0g of 3.5% liquid) into a centrifuge tube, add 8ml of cold (4°C) 0.25mol / lHClO4 into the centrifuge tube, and shake evenly; immediately put the centrifuge tube into a 4°C cold water water bath, keep warm for 15 minutes, do not shake during this period; centrifuge at 4000rpm for 10 minutes, gently pour out the clear liquid on the surface; add 5ml of 0.5mol / l HClO4 to the precipitate, shake and mix; put the centrifuge tube into a 70°C water bath, keep warm for 15 minutes, shake once every 3-4 minutes; centrifuge at 4000rpm for 10 minutes, draw 1ml of supernatant, make up to 100ml with distilled water, and mix evenly.

[0068] 2.3.2 Determination

[0069] Rinse the cuvette with the sample to be tested, fill the cuvette and place it in the spectrophotometer. Wipe the surface clean. Measure the absorbance at 260nm, using distilled water as a blank. Record the absorbance, repeat the measurement once, and take the average of the two measurements.

[0070] 2.3.3 Calculation

[0071]

[0072] Where:

[0073] A——absorbance of sample solution;

[0074] Dilution - When operating according to this method, the dilution is 100%;

[0075] m——the mass of the sample weighed, mg;

[0076] Ds——dry matter content of sample, %;

[0077] 5——The volume of the solution after adding 0.5 mol / L HClO4;

[0078] 0.03365——corresponds to the RNA content in the test solution when the absorbance is 1.00, mg / 100mL.

[0079] 2.3.4 Precision

[0080] The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 3% of the arithmetic mean.

[0081] 3) Determination of reducing sugar

[0082] The determination was carried out according to the first method direct titration method in GB 5009.7-2016 National Food Safety Standard Determination of Reducing Sugar in Food.

[0083] Example 1

[0084] (I) Preparation of biomass saccharification solution and preparation of mixed solution

[0085] 1kg1-10cm sugarcane bagasse raw material is moistened with water, and the moisture content is controlled to 30%, and then transferred to the continuous steam explosion equipment for explosion treatment, wherein the continuous steam explosion pressure is 1.6Mpa, and the high temperature cooking is 10min. After the reaction is completed, the pressure is quickly released, and the slag-liquid mixture after steam explosion is added to the enzymatic hydrolysis reaction tank, and the slag-liquid mixture after steam explosion is prepared into a mixed liquid with a solid content of 18wt% in the reaction tank with 50℃ hot water, and then 1wt% of cellulase (based on the dry matter mass of the mixed liquid) is added, and the pH of the mixed liquid is adjusted to 5.0, and enzymolysis is carried out at 50℃ for 24h; after the enzymolysis is completed, the enzymolysis material is put into a horizontal screw centrifuge, and the supernatant is taken after centrifugation and the supernatant is heated to 65℃, and then plate and frame filter is performed to remove the insoluble matter in the sugar liquid to obtain the biomass saccharification liquid; 3kg of biomass saccharification liquid is taken and copper sulfate, zinc sulfate, ferrous sulfate, magnesium chloride, potassium chloride, calcium chloride, vitamin B1, vitamin Vitamin B2, vitamin B3, vitamin B5, vitamin B6 and biotin are prepared into a mixed solution, in which the mass content of copper element is 5ppm, the mass content of zinc element is 100ppm, the mass content of iron element is 100ppm, the mass content of magnesium element is 10g / kg, the mass content of potassium element is 10g / kg, the mass content of calcium element is 10g / kg, the mass content of vitamin B1 is 50ppm, the mass content of vitamin B2 is 3ppm, the mass content of vitamin B3 is 50ppm, the mass content of vitamin B5 is 50ppm, the mass content of vitamin B6 is 50ppm, the mass content of biotin is 100ppm, and the reducing sugar concentration is 8wt%.

[0086] (II) Production of Yeast Protein

[0087] 1) Slant culture: inoculate the preserved Candida utilis 31170 into a sucrose culture medium, adjust the pH to 4.8, and culture at 30° C. for 24 h; the composition of the culture medium is sucrose 10 wt%, yeast extract 2 wt%, magnesium sulfate 1 wt%, ammonium dihydrogen phosphate 1 wt%, and the rest is water;

[0088] 2) Liquid seed culture: The yeast strain cultured on the slant was inoculated into a special culture bottle containing 1000 mL, inoculated into a sucrose medium, adjusted to pH 4.8, and cultured at 30°C for 24 hours; the composition of the medium was sucrose 10wt%, yeast extract 2wt%, magnesium sulfate 1wt%, ammonium dihydrogen phosphate 1wt%, and the rest was water;

[0089] 3) Fermentation tank seed culture: 2L of liquid seed culture was inoculated into a 45L fermentation tank filled with 18L of bottom water, the pH was adjusted to 4.5, and fermentation was started, wherein the carbon source, nitrogen source, phosphorus source and 15wt% soda ash used for adjusting the pH were all added by flow addition, the carbon source was 30% concentration molasses, the flow acceleration was 0.125-0.2L / h, the nitrogen source was ammonium sulfate, the flow acceleration was 8-20g / h, the phosphorus source was ammonium phosphate, the flow acceleration was 2-4g / h, the fermentation was carried out at 28°C for 24h, and the yeast strain concentrate was collected by centrifugation for subsequent fermentation research.

[0090] 4) Continuous culture in a fermentation tank: 2 L of the yeast concentrate prepared in step 3) was inoculated into a fermentation tank containing 18 L of bottom water, the fermentation mass was controlled to be about 20 kg, the wet weight of the fermented bacteria was 150 g / L, and the yeast was activated for 2 hours before entering into continuous fermentation. The mixed solution, nitrogen source and phosphorus source were added in a flow-addition manner during continuous fermentation. After the fermentation process stabilized, the flow acceleration of the mixed solution was 4-5 kg / h, the flow acceleration of the nitrogen source and phosphorus source was 0.5-0.8 kg / h, and the discharge rate was 4.5-5.5 kg / h. At this time, the specific growth rate of Candida albicans produced in the fermentation process was 0.21 h -1 , fermented at 28℃ for 96h; wherein, ammonia water was used as the nitrogen source, and ammonium phosphate was used as the phosphorus source.

[0091] 5) Separation and drying: separation by a stacked disc separator and plate and frame filter press, and then freeze-drying the filter cake to obtain yeast protein. The absolute dry yield of yeast protein is 15 kg, and the production efficiency is 0.156 kg / h. The protein content and RNA content in the yeast protein are tested every 3 hours during the production process. The protein content in the yeast protein is 52-55wt%, and the RNA content is 8-9wt%.

[0092] Example 2

[0093] (I) Preparation of biomass saccharification solution and preparation of mixed solution

[0094] 1kg1-10cm corn stalks are moistened with water, and the moisture content is controlled at 40%, and then transferred to the instantaneous ejection steam explosion equipment for explosion treatment, wherein the instantaneous ejection steam explosion controls the pressure to 1.2Mpa and the reaction time is 6min. After the reaction is completed, the pressure is quickly released, and the slag-liquid mixture after steam explosion is added to the reaction tank, and the slag-liquid mixture after steam explosion is prepared into a mixed liquid with a solid content of 15wt% in the reaction tank with 50℃ hot water, and 4wt% of cellulase (based on the dry matter mass of the mixed liquid) is added, and the pH is adjusted to 6.0, and enzymolysis is carried out at 60℃ for 48h. After the enzymolysis is completed, the material enters the horizontal screw centrifuge, and the supernatant is taken after centrifugation and heated to 65℃, and then plate and frame filter is performed to remove the insoluble matter in the sugar liquid to obtain the biomass saccharification liquid; 3kg of biomass saccharification liquid is taken and copper sulfate, zinc sulfate, ferrous sulfate, magnesium chloride, potassium chloride, calcium chloride, vitamin B1, vitamin B 2. Vitamin B3, vitamin B5, vitamin B6 and biotin are prepared into a mixed solution, in which the mass content of copper element is 4 ppm, the mass content of zinc element is 60 ppm, the mass content of iron element is 80 ppm, the mass content of magnesium element is 5 g / kg, the mass content of potassium element is 10 g / kg, the mass content of calcium element is 2 g / kg, the mass content of vitamin B1 is 40 ppm, the mass content of vitamin B2 is 3 ppm, the mass content of vitamin B3 is 40 ppm, the mass content of vitamin B5 is 90 ppm, the mass content of vitamin B6 is 40 ppm, the mass content of biotin is 50 ppm, and the reducing sugar concentration is 6%.

[0095] (II) Production of Yeast Protein

[0096] 1) using the method of step 1) to step 3) in Example 1 to perform multi-stage seed culture on Candida utilis 31170 to obtain a yeast strain concentrate;

[0097] 2) Continuous culture in a fermentation tank: 2L of yeast strain concentrate prepared in step 1) is inoculated into a fermentation tank containing 18L of bottom water, the fermentation mass is controlled to be about 20kg, the wet weight of the fermented bacteria is 100g / L, and the yeast is activated for 2h before continuous fermentation. The mixed solution, nitrogen source and phosphorus source are added in a flow-addition manner during continuous fermentation. After the fermentation process is stable, the flow acceleration of the mixed solution is 7-8kg / h, the flow acceleration of the nitrogen source and the phosphorus source is 0.5-0.8kg / h, and the discharge rate is 7.5-8.5kg / h. At this time, the specific growth rate of Candida albicans produced in the fermentation process is 0.32h ～1 , fermented at 30°C for 96h; wherein the nitrogen source is ammonia water and the phosphorus source is ammonium phosphate;

[0098] 3) Separation and drying: separation by a stacked disc separator and plate and frame filter press, and then freeze-drying the filter cake to obtain yeast protein. The absolute dry yield of yeast protein is 13.5 kg, and the production efficiency is 0.14 kg / h. The protein content and RNA content in the yeast protein are tested every 3 hours during the production process. The protein content in the yeast protein is 54-56wt%, and the RNA content is 9-10wt%.

[0099] Example 3

[0100] (I) Preparation of biomass saccharification solution and preparation of mixed solution

[0101] 1kg1-10cm bagasse is moistened with water, the moisture content is controlled at 35%, and then transferred to the instantaneous ejection steam explosion equipment for explosion treatment, wherein the instantaneous ejection steam explosion controls the pressure to 1.0Mpa and the reaction time is 4min. After the reaction is completed, the pressure is quickly released, and the residue-liquid mixture after steam explosion is added to the reaction tank, and the residue-liquid mixture after steam explosion is prepared into a mixed liquid with a solid content of 10wt% in the reaction tank with hot water, and 2wt% of cellulase (based on the dry matter mass of the mixed liquid) is added, and the pH is adjusted to 5.5, and enzymolysis is carried out at 40°C for 96h. After the enzymolysis is completed, the material enters the horizontal screw centrifuge, and the supernatant is taken after centrifugation and heated to 65°C, and then plate and frame filter is performed to remove the insoluble matter in the sugar solution to obtain the biomass saccharification liquid, and 4kg of biomass saccharification liquid is taken and copper sulfate, zinc sulfate, ferrous sulfate, magnesium chloride, potassium chloride, calcium chloride, vitamins B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6 and biotin are prepared into a mixed solution, wherein the mass content of copper element is 3ppm, the mass content of zinc element is 50ppm, the mass content of iron element is 50ppm, the mass content of magnesium element is 2g / kg, the mass content of potassium element is 2g / kg, the mass content of calcium element is 2g / kg, the mass content of vitamin B2 is 1ppm, the mass content of vitamin B3 is 10ppm, the mass content of vitamin B5 is 50ppm, the mass content of vitamin B6 is 10ppm, the mass content of biotin is 10ppm, and the reducing sugar concentration is 3%.

[0102] (II) Production of Yeast Protein

[0103] 1) using the method in Example 1 to perform multi-stage seed culture on Candida utilis 31170 to obtain a yeast strain concentrate;

[0104] 2) Fermentation tank culture: 2L of yeast strains were inoculated into a fermentation tank containing 18L of bottom water, the fermentation mass was controlled to be about 20kg, the wet weight of the fermented bacteria was 50g / L, and the yeast was activated for 2h before continuous fermentation. The mixed solution, nitrogen source and phosphorus source were added in a flow-addition manner during continuous fermentation. After the fermentation process stabilized, the flow acceleration of the mixed solution was 8.5-9.5kg / h, the flow acceleration of the nitrogen source and phosphorus source was 0.5-0.8kg / h, and the discharge rate was 9-10kg / h. At this time, the specific growth rate of Candida albicans produced in the fermentation process was 0.4h -1 , fermentation temperature 32 ℃ 96h; wherein, the nitrogen source is ammonia water, and the phosphorus source is ammonium phosphate.

[0105] 3) Separation and drying: separation by a stacked disc separator and plate and frame filter press, and then freeze-drying the filter cake to obtain yeast protein. The absolute dry yield of yeast protein is 10.5 kg, and the production efficiency is 0.11 kg / h. The protein content and RNA content in the yeast protein are tested every 3 hours during the production process. The protein content in the yeast protein is 56-58wt%, and the RNA content is 11-12wt%.

[0106] Example 4

[0107] The difference from Example 1 is that in Example 4, biomass saccharification liquid is directly fed during continuous fermentation, and copper sulfate, zinc sulfate, ferrous sulfate, magnesium chloride, potassium chloride, calcium chloride, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6 and biotin are not added to the biomass saccharification liquid.

[0108] 1) using the method in Example 1 to perform multi-stage seed culture on Candida utilis 31170 to obtain a yeast strain concentrate;

[0109] 2) Continuous culture in fermentation tank: 2L yeast strains were inoculated into a fermentation tank containing 18L bottom water, the fermentation mass was controlled at about 20kg, the wet weight of the fermented bacteria was 150g / L, and the yeast was activated for 2h before continuous fermentation. Biomass saccharification liquid, nitrogen source and phosphorus source were added by flow addition in continuous fermentation. The initial flow acceleration of 8% biomass saccharification liquid was 4-5kg / h, the flow acceleration of nitrogen source and phosphorus source was 0.5-0.8kg / h, and the discharge rate was 4.5-5.5kg / h. Ammonia water was used as the nitrogen source and ammonium phosphate was used as the phosphorus source. The stable state can be maintained normally in the initial stage of fermentation, but it is difficult to maintain after 6h of fermentation. The wet weight of fermentation is continuously reduced. The fermentation is terminated after 12-18h of continuous fermentation. The wet weight of fermentation decreases by more than 50%, the production efficiency is low, and the sugar conversion efficiency is low. The specific growth rate of Candida albicans during the fermentation process at this stage is calculated to be 0.1-0.15h -1 Yeast milk was collected to test yeast protein product indicators, among which the protein content and RNA content were unstable, the protein content was ≤40%, and the RNA content was ≤6%.

[0110] Due to the abnormal fermentation process and short fermentation time, the production efficiency cannot be calculated normally.

[0111] Comparative Example 1

[0112] The difference from Example 1 is that in Comparative Example 1, molasses is used as a carbon source during continuous fermentation, wherein the reducing sugar concentration of the molasses is 8%, and various trace elements are not added.

[0113] The method in Example 1 was used to perform multi-stage seed culture on Candida utilis 31170 to obtain a yeast strain concentrate;

[0114] Continuous culture in fermentation tank: 2L yeast strains were inoculated into a fermentation tank containing 18L bottom water, the fermentation mass was controlled at about 20kg, the wet weight of the fermented bacteria was 150g / L, and the yeast was activated for 2h before continuous fermentation. 8% concentration of molasses, nitrogen source and phosphorus source were added in a flow-addition manner for continuous fermentation. After the fermentation process stabilized, the molasses flow acceleration was 4-5kg / h, the nitrogen source and phosphorus source flow acceleration were 0.5-0.8kg / h, and the discharge rate was 4.5-5.5kg / h. At this time, the fermentation process had a specific growth rate of 0.21h ～1 About 200 mL of water was added and fermented at 28°C for 96 h. Ammonia water was used as the nitrogen source and ammonium phosphate was used as the phosphorus source.

[0115] Separation and drying: separation by stacked disc separator and plate and frame filter press, and then freeze-drying the filter cake to obtain yeast protein. The absolute dry yield of yeast protein is 14kg, and the production efficiency is 0.146kg / h. The protein content and RNA content in the yeast protein are tested every 3 hours during the production process. The protein content in the yeast protein is 52-54wt%, and the RNA content is 8-9wt%.

[0116] The protein content of the yeast protein obtained in Examples 1 to 3 of the present invention is 52 to 58%, the RNA content is 8 to 12%, the fermentation efficiency is 0.11 to 0.156 kg / h, and the specific growth rate of Candida albicans is 0.21 to 0.4 h ～1 ; It shows that the method of the present invention produces yeast protein, the fermentation process is stable, the production efficiency is high, and the product quality is stable. Compared with Example 1, no nutrients are added to the biomass saccharification liquid in Example 4, the production efficiency is low, the sugar conversion efficiency is low, and normal and stable production cannot be achieved. The protein content and RNA content in the produced yeast protein are unstable; Compared with Example 1, Comparative Example 1 uses molasses as the fermentation carbon source, and the protein content and RNA content in the obtained yeast protein are 52-54%, the RNA content is 8-9%, the fermentation efficiency is 0.146kg / h, and the specific growth rate of Candida albicans is 0.21h -1 The method for preparing yeast protein of the present invention is equivalent to the method for preparing yeast protein using molasses as a fermentation carbon source in terms of production efficiency and product quality.

[0117] In summary, the method of the present invention is used to produce yeast protein, and the fermentation process is stable, the production efficiency is high, and the quality of the produced products is stable. Compared with the method of producing yeast using molasses in the prior art, the method of the present invention is equivalent to the prior art in terms of production efficiency and product quality. Therefore, the method of the present invention can realize the industrial application of preparing yeast protein from low-concentration biomass saccharification liquid, increase the channels for obtaining yeast protein sugar resources, broaden the application scope of low-concentration biomass saccharification liquid, and at the same time alleviate the problem of shortage of molasses resources in the yeast industry, and also increase the application scope of biomass saccharification liquid.

[0118] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing yeast protein using low-concentration biomass saccharification liquid, characterized in that: The following steps are involved: S1, steam explosion and saccharification of biomass to obtain biomass saccharification liquid, wherein the reducing sugar concentration in the biomass saccharification liquid is 2-9wt%, and the biomass is lignocellulosic biomass; the lignocellulosic biomass is selected from one or more of bagasse, straw, corn cob and wood chips; S2. The biomass saccharification liquid and nutrients are mixed into a mixed solution, wherein the nutrients include trace elements and vitamins; based on the mass of the mixed solution, the nutrients include 3-5 ppm copper, 50-100 ppm zinc, 50-100 ppm iron, 2000-10000 ppm potassium, 2000-10000 ppm calcium, 2000-10000 ppm magnesium, 0-50 ppm vitamin B1, 1-3 ppm vitamin B2, 10-50 ppm vitamin B3, 50-100 ppm vitamin B5, 10-50 ppm vitamin B6 and 10-100 ppm biotin; S3, using the mixed solution obtained in step S2 as a carbon source to continuously ferment the yeast strain to obtain yeast protein, and adding a nitrogen source and a phosphorus source during the fermentation process, wherein the yeast strain is Candida albicans.

2. The method according to claim 1, characterized in that The steam explosion is continuous steam explosion or instantaneous ejection steam explosion.

3. The method according to claim 2, characterized in that The pressure of the continuous steam explosion is 1.0-1.6 MPa, or the pressure of the instantaneous ejection steam explosion is 1.0-1.4 MPa.

4. The method according to claim 3, characterized in that The cooking time of continuous steam explosion is 5 to 10 minutes; or, the cooking time of instantaneous ejection steam explosion is 2 to 6 minutes.

5. The method according to claim 1, characterized in that The saccharification treatment uses hemicellulase and cellulase, or uses cellulase for enzymatic hydrolysis.

6. The method according to claim 5, characterized in that The enzyme is added in an amount of 1-4% based on the dry weight of the material after steam explosion.

7. The method according to claim 6, characterized in that The enzyme activity of the enzyme is ≥10000 U / g.

8. The method according to claim 6, characterized in that The enzymatic hydrolysis pH is 5.0-6.0, and the enzymatic hydrolysis temperature is 40-60°C.

9. The method according to claim 8, characterized in that The enzymatic hydrolysis time is 24 to 72 hours.

10. The method according to claim 2, characterized in that The saccharification treatment uses hemicellulase and cellulase, or uses cellulase for enzymatic hydrolysis.

11. The method according to claim 1, characterized in that: After the saccharification treatment, the process also includes a step of separating the biomass saccharification liquid.

12. The method according to claim 2, characterized in that: After the saccharification treatment, the process also includes a step of separating the biomass saccharification liquid.

13. The method according to claim 5, characterized in that After the saccharification treatment, the process also includes a step of separating the biomass saccharification liquid.

14. The method according to claim 1, characterized in that The fermentation temperature is 28-32℃.

15. The method according to claim 14, characterized in that The fermentation pH is 4.5-5.

5.

16. The method according to claim 15, characterized in that The fermentation time is 72 to 120 hours.

17. The method according to claim 2, characterized in that The fermentation temperature is 28-32℃.

18. The method according to claim 5, characterized in that The fermentation temperature is 28-32℃.

19. The method according to claim 11, characterized in that The fermentation temperature is 28-32℃.

20. The method according to any one of claims 1 to 19, characterized in that: Step S3: the fermentation nitrogen source is selected from one or more of urea, ammonium sulfate, ammonia water or yeast extract, and / or, In step (3), the phosphorus source for fermentation is selected from one or more of ammonium phosphate, monoammonium hydrogen phosphate, diammonium hydrogen phosphate and phosphoric acid.

21. The method according to claim 20, characterized in that The flow acceleration of the mixed solution in step S2 is 4 to 10 kg / h.

22. The method according to claim 21, characterized in that The flow acceleration of the fermentation nitrogen source is 0.5-0.8 kg / h, and the flow acceleration of the fermentation phosphorus source is 0.5-0.8 kg / h.

23. The method according to claim 22, characterized in that The specific growth rate of yeast strains during the fermentation process is 0.2-0.4h -1 .

24. The method according to any one of claims 1 to 19, characterized in that: The straw is selected from one or more of rice straw, cotton straw and wheat straw.

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