A new process for purifying β-1,3-glucan from fermentation broth of Saccharomyces cerevisiae cells

Abstract

The present invention belongs to the field of bioengineering, and specifically discloses a new process for purifying β-1,3-glucan from a fermentation broth of a saccharomyces cerevisiae cell; comprising the following steps: 1) preparation of a fermentation broth of saccharomyces cerevisiae, 2) purification of β-1,3-glucan; in the preparation of the fermentation broth of saccharomyces cerevisiae, the following initial culture medium is used for fermentation tank culture, including: 25-35 g / L of glucose, 5-6.5 g / L of ammonium sulfate, 7-12 g / L of yeast extract powder, 5-8 g / L of arginine, 0.4-0.6 g / L of magnesium sulfate, 4.1-5.1 g / L of disodium hydrogen phosphate, and 0.5-0.7 g / L of potassium dihydrogen phosphate. The new process disclosed in the present invention has few steps, high unit yield of β-1,3-glucan, convenient purification, low preparation cost, and obvious industrial value.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically discloses a new process for purifying beta-1,3-glucan from brewer's yeast cell fermentation liquid. Background Art

[0002] β-1,3 glucan is a kind of glucose polymer with multiple biological activities connected by β-1,3 glucan. Its efficacy in application experiments is as follows: moisturizing, anti-inflammatory, anti-aging, anti-wrinkle, anti-dandruff, anti-yellowing, accelerated repair, increased skin elasticity, auxiliary antibacterial, enhanced immunity, auxiliary blood sugar and blood lipid reduction, improved gastrointestinal function, etc. It is widely used in many fields such as medicine, food, cosmetics, animal feed additives, etc.

[0003] Specifically, β-1,3 glucan can activate macrophages and neutrophils, etc., thus increasing the levels of leukocytes, cytokines and special antibodies, and fully stimulating the body's immune system. Then, the body is more prepared to resist diseases caused by microorganisms. β-glucan can quickly restore the ability of injured lymphocytes to produce cytokines (IL-1) to normal, effectively regulating the body's immune function. A large number of experiments have shown that β-glucan can promote the production of IgM antibodies in the body to improve humoral immunity. This type of glucan activates cells and stimulates the host's non-specific defense mechanism, so it has attracted much attention in the application of tumors, infectious diseases and trauma treatment. β-1,3 glucan, which is extracted through special steps and does not contain endotoxin, has been recognized by the US FDA as a safe substance and can be added to general foods. Many reports show that oral administration of yeast β-1,3 glucan to mice can increase the antibacterial phagocytosis of peritoneal cells.

[0004] The cell walls of yeast and other fungi contain a large amount of β-1,3 glucan, so the current β-1,3 glucan is mainly prepared by yeast, such as extracting waste yeast from the beer industry. For example, CN201010214448.5 discloses a method for simultaneously preparing yeast extract and β-1,3-glucan, which includes debittering and removing impurities from waste beer yeast, enzymatic hydrolysis and inactivation of waste beer yeast, separation and drying of yeast extract, acid-base treatment, washing and drying of β-1,3-glucan, etc. This method has the characteristics of high raw material utilization, good quality, low cost, and easy industrialization, and achieves the purpose of reusing waste beer yeast and simultaneously producing two products, yeast extract and β-1,3-glucan. CN200910039693.4 discloses a method for preparing yeast β-1,3-glucan, which uses beer yeast residue liquid or dried beer yeast residue powder, which is a byproduct in the beer production process, as a raw material to prepare high-purity yeast β-1,3-glucan. The preparation method of the present invention is achieved by the following steps: (1) dispersing beer yeast residue in water so that the mass ratio of solids to water is 1:5 to 1:20, and stirring to fully mix; (2) adding α-amylase to the beer yeast residue suspension at 90 to 105°C, stirring, and fully reacting; (3) adding alkali to the suspension at a temperature of 90 to 105°C, stirring, and after sufficient reaction, centrifuging to collect the precipitate; (4) adding water to the precipitate, washing with water to desalinate, and centrifuging to dry. Although the above method of extracting β-1,3 glucan using waste yeast can save the fermentation step, since waste brewing yeast is used, the yeast cells are in poor condition, cell autolysis is common, and there are many impurities in the raw materials, which affects the subsequent separation and purification and greatly affects the purity of β-1,3 glucan.

[0005] CN201510159347.5 discloses a culture medium for improving the content of β-glucan in space-induced yeast strains and its application. The raw material composition and content of the culture medium are: 3.45-3.70g glucose, 3.60-3.95g corn steep liquor, 2.80-3.10g yeast extract, 0.45-0.70g glycerol, and water is supplemented to 100mL; the culture conditions are: 4% (v / v) inoculation amount is accessed into a 50L fermentation tank, the culture medium is filled with the above culture medium, 26-28°C, 450r / min shaking culture for 72h. The cell wall of Saccharomyces cerevisiae is used as raw material, and the carbon source, nitrogen source, inorganic ion and enzyme activator in the culture medium are optimized by single factor experiment and response surface analysis, and the mutant strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) FFLM2.0016 is cultured in a fermentation tank to improve the content of β-glucan.

[0006] However, the above fermentation process requires a specific mutagenic strain, and the yield of β-1,3 glucan is less than 5g / L, resulting in high costs and lack of market competitiveness. Therefore, it is necessary to find a new brewer's yeast fermentation process to increase the yield of β-1,3 glucan, and accordingly find a suitable purification method to achieve an industrially feasible production process. Summary of the invention

[0007] The purpose of the present invention is to provide a new process for purifying β-1,3-glucan from the fermentation broth of Saccharomyces cerevisiae cells, so as to solve the problems raised in the above background technology.

[0008] The present invention adopts the following technical solutions:

[0009] A new process for purifying β-1,3-glucan from a fermentation broth of a yeast cell, comprising the following steps: 1) preparing a fermentation broth of the yeast cell, 2) purifying the β-1,3-glucan;

[0010] In the preparation of the 1) Saccharomyces cerevisiae fermentation broth, the following initial culture medium is used for fermentation tank culture, including:

[0011]

[0012] Furthermore, in the above-mentioned novel process for purifying β-1,3-glucan from the fermentation broth of Saccharomyces cerevisiae cells, the yeast inoculation amount of the initial culture medium in the preparation of the fermentation broth of Saccharomyces cerevisiae cells is 2-6×10 6 Pieces / mL.

[0013] Furthermore, in the above-mentioned new process for purifying β-1,3-glucan from the fermentation broth of Saccharomyces cerevisiae cells, the fermentation tank is a 30L fermentation tank.

[0014] Furthermore, in the above-mentioned novel process for purifying β-1,3-glucan from the fermentation broth of Saccharomyces cerevisiae cells, the 1) preparation of the fermentation broth of Saccharomyces cerevisiae comprises the following specific steps:

[0015] 11) Preparation of seed liquid

[0016] The preserved glycerol strain of Saccharomyces cerevisiae was streaked onto a malt extract medium plate and cultured in a 28-30°C incubator until a single colony grew;

[0017] Pick out a full single colony from the above single colony and streak it on a malt juice medium plate, culture it in a 28-30° C. incubator for 1-2 days, then pick out a full strain and inoculate it into a liquid malt juice medium, culture it in a shaking bottle at 28-30° C. and 200-250 r / min overnight to obtain a seed solution;

[0018] 12) Fermentation in a fermenter

[0019] The seed liquid prepared in step 11) is inoculated into the initial culture medium at an inoculation rate of 11-13% to start fermentation culture, the fermentation culture adopts 31-33° C., stirring and aerating 100-150 r / min; sugar supplementation starts from 10 hours of fermentation culture, and the sugar supplement adopts a pre-prepared sucrose solution with a concentration of 200 g / mL, and the sugar is supplemented once every 12 hours, and 1.50-2.0 mL / L is supplemented each time; the pH value is controlled at 6.8-7.2 for 0-24 hours of fermentation; after 24-48 hours, it is controlled at 6.7-6.9; in the final stage, it is controlled at above 7.0, and the fermentation is terminated after 60-72 hours of fermentation culture.

[0020] Furthermore, the novel process for purifying β-1,3-glucan from the fermentation broth of Saccharomyces cerevisiae cells is characterized in that the purification of β-1,3-glucan comprises the following steps:

[0021] 21) centrifuging the fermented yeast broth at 1000-2000 g to separate the yeast mud;

[0022] 22) preparing a suspension of the yeast paste obtained in step 21) with deionized water, wherein the weight percentage of the suspension is 25-30% based on the dry weight of the yeast, adjusting the pH to 5.5-6.5, and then adding papain and bromelain to the suspension, incubating at 40-65° C. for 16-24 hours to inactivate the enzymes, and centrifuging to obtain a supernatant and a precipitate;

[0023] 23) adding 1 to 4 times the weight of the precipitate to the precipitate obtained in step 22), wherein the weight percentage concentration of the precipitate is 1 to 5%, and reacting at 60 to 90° C. for 2 to 4 hours, cooling to room temperature, centrifuging at 1500-2000 g, removing the supernatant, and retaining the precipitate;

[0024] 24) adding 0.2-0.4 mol / L acetic acid solution to the precipitate obtained in step 23) to suspend it, wherein the mass volume ratio of the precipitate to the acetic acid solution is 1:5, treating at 65-70° C. for 1 h, cooling to room temperature and centrifuging, washing the precipitate to obtain muddy β-1,3-glucan;

[0025] 25) The sludge-like β-1,3-glucan obtained in step 24) is subjected to vacuum freeze-drying for 25 to 35 hours to obtain powdered β-1,3-glucan.

[0026] Furthermore, in the above-mentioned new process for purifying β-1,3-glucan from the fermentation broth of Saccharomyces cerevisiae cells, the amount of papain added in step 22) is 1500-1800 U / L, and the amount of bromelain added is 500-1000 U / L.

[0027] Furthermore, in the above-mentioned new process for purifying β-1,3-glucan from the fermentation broth of Saccharomyces cerevisiae cells, the alkali in step 23) is sodium hydroxide.

[0028] Furthermore, the present invention also discloses a β-1,3-glucan prepared by the new process described in any one of claims 1-7.

[0029] Furthermore, the present invention also discloses the use of the above-mentioned β-1,3-glucan or its preparation process in the preparation of food additives or medicines.

[0030] The present invention has the following beneficial effects:

[0031] The invention discloses a new process for purifying beta-1,3-glucan from a fermentation liquid of a saccharomyces cerevisiae cell. A certain amount of arginine is added to an initial culture medium in a fermentation stage, and it is found that the unit yield of the beta-1,3-glucan can be greatly promoted. The possible reason is that arginine participates in the activation of the activity of key enzymes in the synthesis pathway of beta-1,3-glucan by saccharomyces cerevisiae. The invention also finds that in the extraction process stage, papain and bromelain are used in combination to hydrolyze yeast cell walls, so that the beta-1,3-glucan can be more thoroughly hydrolyzed, and other impurities can also be more thoroughly decomposed and more easily removed in post-treatment, thereby improving the purity and yield of the beta-1,3-glucan. In summary, the process disclosed in the present invention is further optimized on the existing technology, using S288C brewer's yeast that is easily available on the market as a fermentation bacteria, using a relatively cheap culture medium, and through a shorter fermentation process and a simple extraction process, a high yield (>8g / L) and purity (above 88%) of β-1,3-glucan are achieved, which is very suitable for industrial production and has good practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Test Example 1: Comparison of β-1,3-glucan production;

[0033] Figure 2 Test Example 1: Comparison of HPLC purity of β-1,3-glucan;

[0034] Figure 3 Test Example 2: Comparison of β-1,3-glucan production;

[0035] Figure 4 Test Example 2: Comparison of HPLC purity of β-1,3-glucan. DETAILED DESCRIPTION

[0036] A new process for purifying β-1,3-glucan from a fermentation broth of a yeast cell, comprising the following steps: 1) preparing a fermentation broth of the yeast cell, 2) purifying the β-1,3-glucan;

[0037] In the preparation of the 1) Saccharomyces cerevisiae fermentation broth, the following initial culture medium is used for fermentation tank culture, including:

[0038]

[0039] In the preparation of the fermentation broth of Saccharomyces cerevisiae, the yeast inoculum amount of the initial culture medium is 2-6×10 6 Pieces / mL;

[0040] The fermentation tank is a 30L fermentation tank;

[0041] The preparation of 1) Saccharomyces cerevisiae fermentation broth comprises the following specific steps:

[0042] 11) Preparation of seed liquid

[0043] The preserved glycerol strain of Saccharomyces cerevisiae was streaked onto a malt extract medium plate and cultured in a 28-30°C incubator until a single colony grew;

[0044] Pick out a full single colony from the above single colony and streak it on a malt juice medium plate, culture it in a 28-30° C. incubator for 1-2 days, then pick out a full strain and inoculate it into a liquid malt juice medium, culture it in a shaking bottle at 28-30° C. and 200-250 r / min overnight to obtain a seed solution;

[0045] 12) Fermentation in a fermenter

[0046] The seed liquid prepared in step 11) is inoculated into the initial culture medium at an inoculum of 11-13% to start fermentation culture, the fermentation culture is carried out at 31-33°C, and the stirring and ventilation are 100-150r / min; sugar supplementation is started from 10h of fermentation culture, and the sugar supplementation uses a pre-prepared sucrose solution with a concentration of 200g / mL, and the sugar is supplemented once every 12h, and 1.50-2.0mL / L is supplemented each time; the pH value is controlled at 6.8-7.2 for 0-24h of fermentation; after 24-48h, it is controlled at 6.7-6.9; in the final stage, it is controlled at above 7.0, and the fermentation is terminated after 60-72h of fermentation culture;

[0047] The purification of 2) β-1,3-glucan comprises the following steps:

[0048] 21) centrifuging the fermented yeast broth at 1000-2000 g to separate the yeast mud;

[0049] 22) preparing a suspension of the yeast paste obtained in step 21) with deionized water, wherein the weight percentage of the suspension is 25-30% based on the dry weight of the yeast, adjusting the pH to 5.5-6.5, and then adding papain and bromelain to the suspension, incubating at 40-65° C. for 16-24 hours to inactivate the enzymes, and centrifuging to obtain a supernatant and a precipitate;

[0050] 23) adding 1 to 4 times the weight of the precipitate to the precipitate obtained in step 22), wherein the weight percentage concentration of the precipitate is 1 to 5%, and reacting at 60 to 90° C. for 2 to 4 hours, cooling to room temperature, centrifuging at 1500-2000 g, removing the supernatant, and retaining the precipitate;

[0051] 24) adding 0.2-0.4 mol / L acetic acid solution to the precipitate obtained in step 23) to suspend the precipitate, wherein the mass volume ratio of the precipitate to the acetic acid solution is 1:5, treating at 65-70° C. for 1 h, cooling to room temperature and centrifuging, washing the precipitate to obtain a sludge-like β-1,3-glucan;

[0052] 25) The sludge-like β-1,3-glucan obtained in step 24) is subjected to vacuum freeze-drying for 25 to 35 hours to obtain powdered β-1,3-glucan.

[0053] In the step 22), the amount of papain added is 1500-1800 U / L, and the amount of bromelain added is 500-1000 U / L;

[0054] The alkali in step 23) is sodium hydroxide.

[0055] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on 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.

[0056] The reagents in the present invention are all purchased from the open market.

[0057] Example 1

[0058] A new process for purifying β-1,3-glucan from a fermentation broth of a yeast cell, comprising the following steps: 1) preparing a fermentation broth of the yeast cell, 2) purifying the β-1,3-glucan;

[0059] In the preparation of the 1) Saccharomyces cerevisiae fermentation broth, the following initial culture medium is used for fermentation tank culture, including:

[0060]

[0061] In the preparation of the brewer's yeast fermentation broth, the yeast inoculation amount of the initial culture medium is 2×10 6 Pieces / mL;

[0062] The fermentation tank is a 30L fermentation tank;

[0063] The preparation of 1) Saccharomyces cerevisiae fermentation broth comprises the following specific steps:

[0064] 11) Preparation of seed liquid

[0065] The preserved glycerol strain of Saccharomyces cerevisiae was streaked onto a malt extract medium plate and cultured in a 28-30°C incubator until a single colony grew;

[0066] Pick out a full single colony from the above single colony and streak it on a malt juice medium plate, culture it in a 28-30° C. incubator for 1-2 days, then pick out a full strain and inoculate it into a liquid malt juice medium, culture it in a shaking bottle at 28-30° C. and 200-250 r / min overnight to obtain a seed solution;

[0067] 12) Fermentation in a fermenter

[0068] The seed liquid prepared in step 11) is inoculated into the initial culture medium at an inoculum of 11-13% to start fermentation culture, the fermentation culture is carried out at 31-33°C, and the stirring and ventilation are 100-150r / min; sugar supplementation is started from 10h of fermentation culture, and the sugar supplementation uses a pre-prepared sucrose solution with a concentration of 200g / mL, and the sugar is supplemented once every 12h, and 1.50-2.0mL / L is supplemented each time; the pH value is controlled at 6.8-7.2 for 0-24h of fermentation; after 24-48h, it is controlled at 6.7-6.9; in the final stage, it is controlled at above 7.0, and the fermentation is terminated after 60-72h of fermentation culture;

[0069] The purification of 2) β-1,3-glucan comprises the following steps:

[0070] 21) centrifuging the fermented yeast broth at 1000-2000 g to separate the yeast mud;

[0071] 22) preparing a suspension of the yeast paste obtained in step 21) with deionized water, wherein the weight percentage of the suspension is 25-30% based on the dry weight of the yeast, adjusting the pH to 5.5, and then adding papain and bromelain to the suspension, incubating at 40° C. for 24 hours to inactivate the enzymes, and centrifuging to obtain a supernatant and a precipitate;

[0072] 23) adding an alkaline solution of 1 times the weight of the precipitate to the precipitate obtained in step 22), wherein the weight percentage concentration of the alkaline solution is 5%, and reacting at 60° C. for 4 hours. After cooling to room temperature, centrifuging at 1500-2000 g, removing the supernatant, and retaining the precipitate;

[0073] 24) adding 0.2 mol / L acetic acid solution to the precipitate obtained in step 23) to suspend it, wherein the mass volume ratio of the precipitate to the acetic acid solution is 1:5, treating at 65-70° C. for 1 h, cooling to room temperature and centrifuging, washing the precipitate to obtain muddy β-1,3-glucan;

[0074] 25) The sludge-like β-1,3-glucan obtained in step 24) is subjected to vacuum freeze-drying for 25 to 35 hours to obtain powdered β-1,3-glucan.

[0075] In the step 22), the amount of papain added is 1500 U / L, and the amount of bromelain added is 500 U / L;

[0076] The alkali in step 23) is sodium hydroxide.

[0077] Example 2

[0078] A new process for purifying β-1,3-glucan from a fermentation broth of a yeast cell, comprising the following steps: 1) preparing a fermentation broth of the yeast cell, 2) purifying the β-1,3-glucan;

[0079] In the preparation of the 1) Saccharomyces cerevisiae fermentation broth, the following initial culture medium is used for fermentation tank culture, including:

[0080]

[0081] In the preparation of the brewer's yeast fermentation broth, the yeast inoculation amount of the initial culture medium is 4×10 6 Pieces / mL;

[0082] The fermentation tank is a 30L fermentation tank;

[0083] The preparation of 1) Saccharomyces cerevisiae fermentation broth comprises the following specific steps:

[0084] 11) Preparation of seed liquid

[0085] The preserved glycerol strain of Saccharomyces cerevisiae was streaked onto a malt extract medium plate and cultured in a 28-30°C incubator until a single colony grew;

[0086] Pick out a full single colony from the above single colony and streak it on a malt juice medium plate, culture it in a 28-30° C. incubator for 1-2 days, then pick out a full strain and inoculate it into a liquid malt juice medium, culture it in a shaking bottle at 28-30° C. and 200-250 r / min overnight to obtain a seed solution;

[0087] 12) Fermentation in a fermenter

[0088] The seed liquid prepared in step 11) is inoculated into the initial culture medium at an inoculum of 11-13% to start fermentation culture, the fermentation culture is carried out at 31-33°C, and the stirring and ventilation are 100-150r / min; sugar supplementation is started from 10h of fermentation culture, and the sugar supplementation uses a pre-prepared sucrose solution with a concentration of 200g / mL, and the sugar is supplemented once every 12h, and 1.50-2.0mL / L is supplemented each time; the pH value is controlled at 6.8-7.2 for 0-24h of fermentation; after 24-48h, it is controlled at 6.7-6.9; in the final stage, it is controlled at above 7.0, and the fermentation is terminated after 60-72h of fermentation culture;

[0089] The purification of 2) β-1,3-glucan comprises the following steps:

[0090] 21) centrifuging the fermented yeast broth at 1000-2000 g to separate the yeast mud;

[0091] 22) preparing a suspension of the yeast paste obtained in step 21) with deionized water, wherein the weight percentage of the suspension is 25-30% based on the dry weight of the yeast, adjusting the pH to 6.0, and then adding papain and bromelain to the suspension, incubating at 55° C. for 20 h to inactivate the enzymes, and centrifuging to obtain a supernatant and a precipitate;

[0092] 23) adding an alkaline solution twice the weight of the precipitate to the precipitate obtained in step 22), wherein the weight percentage concentration of the alkaline solution is 3%, and reacting at 75° C. for 3 h. After cooling to room temperature, centrifuging at 1500-2000 g, removing the supernatant, and retaining the precipitate;

[0093] 24) adding 0.3 mol / L acetic acid solution to the precipitate obtained in step 23) to suspend it, wherein the mass volume ratio of the precipitate to the acetic acid solution is 1:5, treating at 65-70° C. for 1 h, cooling to room temperature and centrifuging, washing the precipitate to obtain muddy β-1,3-glucan;

[0094] 25) The sludge-like β-1,3-glucan obtained in step 24) is subjected to vacuum freeze-drying for 25 to 35 hours to obtain powdered β-1,3-glucan.

[0095] In the step 22), the amount of papain added is 1600 U / L, and the amount of bromelain added is 800 U / L;

[0096] The alkali in step 23) is sodium hydroxide.

[0097] Example 3

[0098] A new process for purifying β-1,3-glucan from a fermentation broth of a yeast cell, comprising the following steps: 1) preparing a fermentation broth of the yeast cell, 2) purifying the β-1,3-glucan;

[0099] In the preparation of the 1) Saccharomyces cerevisiae fermentation broth, the following initial culture medium is used for fermentation tank culture, including:

[0100]

[0101]

[0102] In the preparation of the brewer's yeast fermentation broth, the yeast inoculation amount of the initial culture medium is 6×10 6 Pieces / mL;

[0103] The fermentation tank is a 30L fermentation tank;

[0104] The preparation of 1) Saccharomyces cerevisiae fermentation broth comprises the following specific steps:

[0105] 11) Preparation of seed liquid

[0106] The preserved glycerol strain of Saccharomyces cerevisiae was streaked onto a malt extract medium plate and cultured in a 28-30°C incubator until a single colony grew;

[0107] Pick out a full single colony from the above single colony and streak it on a malt juice medium plate, culture it in a 28-30° C. incubator for 1-2 days, then pick out a full strain and inoculate it into a liquid malt juice medium, culture it in a shaking bottle at 28-30° C. and 200-250 r / min overnight to obtain a seed solution;

[0108] 12) Fermentation in a fermenter

[0109] The seed liquid prepared in step 11) is inoculated into the initial culture medium at an inoculum of 11-13% to start fermentation culture, the fermentation culture is carried out at 31-33°C, and the stirring and ventilation are 100-150r / min; sugar supplementation is started from 10h of fermentation culture, and the sugar supplementation uses a pre-prepared sucrose solution with a concentration of 200g / mL, and the sugar is supplemented once every 12h, and 1.50-2.0mL / L is supplemented each time; the pH value is controlled at 6.8-7.2 for 0-24h of fermentation; after 24-48h, it is controlled at 6.7-6.9; in the final stage, it is controlled at above 7.0, and the fermentation is terminated after 60-72h of fermentation culture;

[0110] The purification of 2) β-1,3-glucan comprises the following steps:

[0111] 21) centrifuging the fermented yeast broth at 1000-2000 g to separate the yeast mud;

[0112] 22) preparing a suspension of the yeast paste obtained in step 21) with deionized water, wherein the weight percentage of the suspension is 25-30% based on the dry weight of the yeast, adjusting the pH to 6.5, and then adding papain and bromelain to the suspension, incubating at 65° C. for 16 h to inactivate the enzymes, and centrifuging to obtain a supernatant and a precipitate;

[0113] 23) adding an alkaline solution 4 times the weight of the precipitate to the precipitate obtained in step 22), wherein the weight percentage concentration of the alkaline solution is 1%, and reacting at 90° C. for 4 hours. After cooling to room temperature, centrifuging at 1500-2000 g, removing the supernatant, and retaining the precipitate;

[0114] 24) adding 0.4 mol / L acetic acid solution to the precipitate obtained in step 23) to suspend it, wherein the mass volume ratio of the precipitate to the acetic acid solution is 1:5, treating at 65-70° C. for 1 h, cooling to room temperature and centrifuging, washing the precipitate to obtain muddy β-1,3-glucan;

[0115] 25) The sludge-like β-1,3-glucan obtained in step 24) is subjected to vacuum freeze-drying for 25 to 35 hours to obtain powdered β-1,3-glucan.

[0116] In the step 22), the amount of papain added is 1800 U / L, and the amount of bromelain added is 1000 U / L;

[0117] The alkali in step 23) is sodium hydroxide.

[0118] Example 4

[0119] A new process for purifying β-1,3-glucan from a fermentation broth of a yeast cell, comprising the following steps: 1) preparing a fermentation broth of the yeast cell, 2) purifying the β-1,3-glucan;

[0120] In the preparation of the 1) Saccharomyces cerevisiae fermentation broth, the following initial culture medium is used for fermentation tank culture, including:

[0121]

[0122] In the preparation of the brewer's yeast fermentation broth, the yeast inoculation amount of the initial culture medium is 4×10 6 Pieces / mL;

[0123] The fermentation tank is a 30L fermentation tank;

[0124] The preparation of 1) Saccharomyces cerevisiae fermentation broth comprises the following specific steps:

[0125] 11) Preparation of seed liquid

[0126] The preserved glycerol strain of Saccharomyces cerevisiae was streaked onto a malt extract medium plate and cultured in a 28-30°C incubator until a single colony grew;

[0127] Pick out a full single colony from the above single colony and streak it on a malt juice medium plate, culture it in a 28-30° C. incubator for 1-2 days, then pick out a full strain and inoculate it into a liquid malt juice medium, culture it in a shaking bottle at 28-30° C. and 200-250 r / min overnight to obtain a seed solution;

[0128] 12) Fermentation in a fermenter

[0129] The seed liquid prepared in step 11) is inoculated into the initial culture medium at an inoculum of 11-13% to start fermentation culture, the fermentation culture is carried out at 31-33°C, and the stirring and ventilation are 100-150r / min; sugar supplementation is started from 10h of fermentation culture, and the sugar supplementation uses a pre-prepared sucrose solution with a concentration of 200g / mL, and the sugar is supplemented once every 12h, and 1.50-2.0mL / L is supplemented each time; the pH value is controlled at 6.8-7.2 for 0-24h of fermentation; after 24-48h, it is controlled at 6.7-6.9; in the final stage, it is controlled at above 7.0, and the fermentation is terminated after 60-72h of fermentation culture;

[0130] The purification of 2) β-1,3-glucan comprises the following steps:

[0131] 21) centrifuging the fermented yeast broth at 1000-2000 g to separate the yeast mud;

[0132] 22) preparing a suspension of the yeast paste obtained in step 21) with deionized water, wherein the weight percentage of the suspension is 25-30% based on the dry weight of the yeast, adjusting the pH to 6.0, and then adding papain to the suspension, incubating at 55° C. for 20 h to inactivate the enzyme, and centrifuging to obtain a supernatant and a precipitate;

[0133] 23) adding an alkaline solution twice the weight of the precipitate to the precipitate obtained in step 22), wherein the weight percentage concentration of the alkaline solution is 3%, and reacting at 75° C. for 3 h. After cooling to room temperature, centrifuging at 1500-2000 g, removing the supernatant, and retaining the precipitate;

[0134] 24) adding 0.3 mol / L acetic acid solution to the precipitate obtained in step 23) to suspend it, wherein the mass volume ratio of the precipitate to the acetic acid solution is 1:5, treating at 65-70° C. for 1 h, cooling to room temperature and centrifuging, washing the precipitate to obtain muddy β-1,3-glucan;

[0135] 25) The sludge-like β-1,3-glucan obtained in step 24) is subjected to vacuum freeze-drying for 25 to 35 hours to obtain powdered β-1,3-glucan.

[0136] The amount of papain added in step 22) is 1600 U / L;

[0137] The alkali in step 23) is sodium hydroxide.

[0138] Compared with Example 2, this example does not add bromelain.

[0139] Example 5

[0140] A new process for purifying β-1,3-glucan from a fermentation broth of a yeast cell, comprising the following steps: 1) preparing a fermentation broth of the yeast cell, 2) purifying the β-1,3-glucan;

[0141] In the preparation of the 1) Saccharomyces cerevisiae fermentation broth, the following initial culture medium is used for fermentation tank culture, including:

[0142]

[0143] In the preparation of the brewer's yeast fermentation broth, the yeast inoculation amount of the initial culture medium is 4×10 6 Pieces / mL;

[0144] The fermentation tank is a 30L fermentation tank;

[0145] The preparation of 1) Saccharomyces cerevisiae fermentation broth comprises the following specific steps:

[0146] 11) Preparation of seed liquid

[0147] The preserved glycerol strain of Saccharomyces cerevisiae was streaked onto a malt extract medium plate and cultured in a 28-30°C incubator until a single colony grew;

[0148] Pick out a full single colony from the above single colony and streak it on a malt juice medium plate, culture it in a 28-30° C. incubator for 1-2 days, then pick out a full strain and inoculate it into a liquid malt juice medium, culture it in a shaking bottle at 28-30° C. and 200-250 r / min overnight to obtain a seed solution;

[0149] 12) Fermentation in a fermenter

[0150] The seed liquid prepared in step 11) is inoculated into the initial culture medium at an inoculum of 11-13% to start fermentation culture, the fermentation culture is carried out at 31-33°C, and the stirring and ventilation are 100-150r / min; sugar supplementation is started from 10h of fermentation culture, and the sugar supplementation uses a pre-prepared sucrose solution with a concentration of 200g / mL, and the sugar is supplemented once every 12h, and 1.50-2.0mL / L is supplemented each time; the pH value is controlled at 6.8-7.2 for 0-24h of fermentation; after 24-48h, it is controlled at 6.7-6.9; in the final stage, it is controlled at above 7.0, and the fermentation is terminated after 60-72h of fermentation culture;

[0151] The purification of 2) β-1,3-glucan comprises the following steps:

[0152] 21) centrifuging the fermented yeast broth at 1000-2000 g to separate the yeast mud;

[0153] 22) preparing a suspension of the yeast paste obtained in step 21) with deionized water, wherein the weight percentage of the suspension is 25-30% based on the dry weight of the yeast, adjusting the pH to 6.0, and then adding bromelain to the suspension, incubating at 55° C. for 20 h to inactivate the enzyme, and centrifuging to obtain a supernatant and a precipitate;

[0154] 23) adding an alkaline solution twice the weight of the precipitate to the precipitate obtained in step 22), wherein the weight percentage concentration of the alkaline solution is 3%, and reacting at 75° C. for 3 h. After cooling to room temperature, centrifuging at 1500-2000 g, removing the supernatant, and retaining the precipitate;

[0155] 24) adding 0.3 mol / L acetic acid solution to the precipitate obtained in step 23) to suspend it, wherein the mass volume ratio of the precipitate to the acetic acid solution is 1:5, treating at 65-70° C. for 1 h, cooling to room temperature and centrifuging, washing the precipitate to obtain muddy β-1,3-glucan;

[0156] 25) The sludge-like β-1,3-glucan obtained in step 24) is subjected to vacuum freeze-drying for 25 to 35 hours to obtain powdered β-1,3-glucan.

[0157] The amount of bromelain added in step 22) is 800 U / L;

[0158] The alkali in step 23) is sodium hydroxide.

[0159] Compared with Example 2, in Example 5, papain was not added.

[0160] Test Example 1

[0161] The yield and purity of the β-1,3-glucan prepared in Examples 1-5 were compared. The yield was calculated based on the dry weight of the purified β-1,3-glucan finally obtained per liter of fermentation broth. The purity was determined using HPLC. The results are shown in Table 1 and the attached Figure 1 and 2 shown.

[0162] Table 1 Test Example 1: Yield and Purity Test

[0163] Yield (g / L) HPLC purity (%) Example 1 8.12 94.62 Example 2 8.46 95.33 Example 3 8.68 95.62 Example 4 7.65 88.71 Example 5 7.39 88.43

[0164] As can be seen from Table 1, as the concentration of the culture medium increases, the yield of β-1,3-glucan also increases. After cultivation using this culture medium, the minimum yield is greater than 8 g / L. At the same time, when papain and bromelain are used together in the re-purification process, the purity of β-1,3-glucan can be increased by more than 5%, reaching more than 94%, which is convenient for subsequent further purification and downstream processing.

[0165] Comparative Example 1

[0166] A new process for purifying β-1,3-glucan from a fermentation broth of a yeast cell, comprising the following steps: 1) preparing a fermentation broth of the yeast cell, 2) purifying the β-1,3-glucan;

[0167] In the preparation of the 1) Saccharomyces cerevisiae fermentation broth, the following initial culture medium is used for fermentation tank culture, including:

[0168]

[0169] In the preparation of the brewer's yeast fermentation broth, the yeast inoculation amount of the initial culture medium is 4×10 6 Pieces / mL;

[0170] The fermentation tank is a 30L fermentation tank;

[0171] The preparation of 1) Saccharomyces cerevisiae fermentation broth comprises the following specific steps:

[0172] 11) Preparation of seed liquid

[0173] The preserved glycerol strain of Saccharomyces cerevisiae was streaked onto a malt extract medium plate and cultured in a 28-30°C incubator until a single colony grew;

[0174] Pick out a full single colony from the above single colony and streak it on a malt juice medium plate, culture it in a 28-30° C. incubator for 1-2 days, then pick out a full strain and inoculate it into a liquid malt juice medium, culture it in a shaking bottle at 28-30° C. and 200-250 r / min overnight to obtain a seed solution;

[0175] 12) Fermentation in a fermenter

[0176] The seed liquid prepared in step 11) is inoculated into the initial culture medium at an inoculum of 11-13% to start fermentation culture, the fermentation culture is carried out at 31-33°C, and the stirring and ventilation are 100-150r / min; sugar supplementation is started from 10h of fermentation culture, and the sugar supplementation uses a pre-prepared sucrose solution with a concentration of 200g / mL, and the sugar is supplemented once every 12h, and 1.50-2.0mL / L is supplemented each time; the pH value is controlled at 6.8-7.2 for 0-24h of fermentation; after 24-48h, it is controlled at 6.7-6.9; in the final stage, it is controlled at above 7.0, and the fermentation is terminated after 60-72h of fermentation culture;

[0177] The purification of 2) β-1,3-glucan comprises the following steps:

[0178] 21) centrifuging the fermented yeast broth at 1000-2000 g to separate the yeast mud;

[0179] 22) preparing a suspension of the yeast paste obtained in step 21) with deionized water, wherein the weight percentage of the suspension is 25-30% based on the dry weight of the yeast, adjusting the pH to 6.0, and then adding papain and bromelain to the suspension, incubating at 55° C. for 20 h to inactivate the enzymes, and centrifuging to obtain a supernatant and a precipitate;

[0180] 23) adding an alkaline solution twice the weight of the precipitate to the precipitate obtained in step 22), wherein the weight percentage concentration of the alkaline solution is 3%, and reacting at 75° C. for 3 h. After cooling to room temperature, centrifuging at 1500-2000 g, removing the supernatant, and retaining the precipitate;

[0181] 24) adding 0.3 mol / L acetic acid solution to the precipitate obtained in step 23) to suspend it, wherein the mass volume ratio of the precipitate to the acetic acid solution is 1:5, treating at 65-70° C. for 1 h, cooling to room temperature and centrifuging, washing the precipitate to obtain muddy β-1,3-glucan;

[0182] 25) The sludge-like β-1,3-glucan obtained in step 24) is subjected to vacuum freeze-drying for 25 to 35 hours to obtain powdered β-1,3-glucan.

[0183] In the step 22), the amount of papain added is 1600 U / L, and the amount of bromelain added is 800 U / L;

[0184] The alkali in step 23) is sodium hydroxide.

[0185] Compared with Example 2, the culture medium in Comparative Example 1 does not contain arginine.

[0186] Comparative Example 2

[0187] Compared with Example 2, the culture medium in this Comparative Example 2 uses the same amount of tyrosine instead of arginine.

[0188] Comparative Example 3

[0189] Compared with Example 2, the culture medium in Comparative Example 3 used the same amount of phenylalanine instead of arginine.

[0190] Comparative Example 4

[0191] Compared with Example 2, the culture medium in Comparative Example 4 used the same amount of lysine instead of arginine.

[0192] Comparative Example 5

[0193] Compared with Example 2, the culture medium in Comparative Example 5 uses the same amount of tryptophan instead of arginine.

[0194] Test Example 2

[0195] The yield and purity of the β-1,3-glucan prepared in Example 2 and Comparative Examples 1-5 were compared. The yield was calculated based on the dry weight of the purified β-1,3-glucan finally obtained per liter of fermentation broth, and the purity was detected by HPLC. The results are shown in Table 2 and Attached Figure 3 and 4 shown.

[0196] Table 2 Test Example 2: Yield and Purity Test

[0197]

[0198]

[0199] As can be seen from Table 2, after adding an appropriate amount of arginine to the culture medium, the production of β-1,3-glucan has been significantly increased. When arginine is replaced by the other four main amino acids, the same effect is not produced. In addition, since the content of β-1,3-glucan is relatively small, the purification effect in the subsequent purification process is not as good as the case of adding arginine.

[0200] It can be seen from the above embodiments, comparative examples and test examples that the present invention discloses a new process for purifying β-1,3-glucan from the fermentation broth of Saccharomyces cerevisiae cells. The present invention adds a certain amount of arginine to the initial culture medium in the fermentation stage, and it is found that the unit yield of β-1,3-glucan can be greatly promoted. The possible reason is that arginine participates in the activation of the activity of key enzymes in the synthesis pathway of β-1,3-glucan by Saccharomyces cerevisiae, and the present invention finds that in the extraction process stage, papain and bromelain are used to jointly hydrolyze the yeast cell wall, so that β-1,3-glucan can be more thoroughly hydrolyzed, and other impurities can also be decomposed more thoroughly and are easier to remove in post-treatment, thereby improving the purity and yield of β-1,3-glucan. In summary, the process disclosed in the present invention is further optimized on the existing technology, using S288C brewer's yeast that is easily available on the market as a fermentation bacteria, using a relatively cheap culture medium, and through a shorter fermentation process and a simple extraction process, a high yield (>8g / L) and purity (above 88%) of β-1,3-glucan are achieved, which is very suitable for industrial production and has good practicality.

[0201] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for purifying β-1,3-glucan from a fermentation broth of Saccharomyces cerevisiae cells, characterized in that: Includes the following Steps: 1) preparation of brewer's yeast fermentation broth, 2) purification of β-1,3-glucan; In the preparation of the 1) Saccharomyces cerevisiae fermentation broth, the following initial culture medium is used for fermentation tank culture, including: Glucose 25-35g / L Ammonium sulfate 5-6.5g / L Yeast extract powder 7-12g / L Arginine 5-8g / L Magnesium sulfate 0.4-0.6g / L Disodium hydrogen phosphate 4.1-5.1g / L Potassium dihydrogen phosphate 0.5-0.7g / L.

2. The method for purifying β-1,3-glucan from a fermentation broth of Saccharomyces cerevisiae cells according to claim 1, characterized in that: In the preparation of the fermentation broth of Saccharomyces cerevisiae, the yeast inoculum amount of the initial culture medium is 2-6×10 6 Pieces / mL.

3. The method for purifying β-1,3-glucan from a fermentation broth of Saccharomyces cerevisiae cells according to claim 1, characterized in that: The fermentation tank is a 30L fermentation tank.

4. The method for purifying β-1,3-glucan from a fermentation broth of Saccharomyces cerevisiae cells according to claim 1, characterized in that: The preparation of 1) Saccharomyces cerevisiae fermentation broth comprises the following specific steps: 11) Preparation of seed liquid Streak the preserved glycerol strain of Saccharomyces cerevisiae on a malt extract medium plate and culture it in a 28-30°C incubator until a single colony grows; Pick out a full single colony from the above single colony and streak it on a malt juice medium plate, culture it in a 28-30° C. incubator for 1-2 days, then pick out a full strain and inoculate it into a liquid malt juice medium, culture it in a shaking bottle at 28-30° C. and 200-250 r / min overnight to obtain a seed solution; 12) Fermentation in a fermenter The seed liquid prepared in step 11) is inoculated into the initial culture medium at an inoculation rate of 11-13% to start fermentation culture, the fermentation culture adopts 31-33° C., stirring and aerating 100-150 r / min; sugar supplementation starts from 10 hours of fermentation culture, and the sugar supplement adopts a pre-prepared sucrose solution with a concentration of 200 g / mL, and the sugar is supplemented once every 12 hours, and 1.50-2.0 mL / L is supplemented each time; the pH value is controlled at 6.8-7.2 for 0-24 hours of fermentation; after 24-48 hours, it is controlled at 6.7-6.9; in the final stage, it is controlled at above 7.0, and the fermentation is terminated after 60-72 hours of fermentation culture.

5. The method for purifying β-1,3-glucan from a fermentation broth of Saccharomyces cerevisiae cells according to claim 1, characterized in that: The purification of 2) β-1,3-glucan comprises the following steps: 21) Centrifuging the fermented yeast broth at 1000-2000 g to separate the yeast paste; 22) preparing a suspension of the yeast paste obtained in step 21) with deionized water, wherein the weight percentage of the suspension is 25-30% based on the dry weight of the yeast, adjusting the pH to 5.5-6.5, and then adding papain and bromelain to the suspension, incubating at 40-65° C. for 16-24 hours to inactivate the enzymes, and centrifuging to obtain a supernatant and a precipitate; 23) adding 1 to 4 times the weight of the precipitate to the precipitate obtained in step 22), wherein the weight percentage concentration of the precipitate is 1 to 5%, and reacting at 60 to 90° C. for 2 to 4 hours, cooling to room temperature, centrifuging at 1500-2000 g, removing the supernatant, and retaining the precipitate; 24) adding 0.2-0.4 mol / L acetic acid solution to the precipitate obtained in step 23) to suspend the precipitate, wherein the mass volume ratio of the precipitate to the acetic acid solution is 1:5, treating at 65-70° C. for 1 h, cooling to room temperature and centrifuging, washing the precipitate to obtain a sludge-like β-1,3-glucan; 25) The sludge-like β-1,3-glucan obtained in step 24) is subjected to vacuum freeze-drying for 25 to 35 hours to obtain powdered β-1,3-glucan.

6. A method for purifying β-1,3-glucan from a fermentation broth of Saccharomyces cerevisiae cells according to claim 5, characterized in that , the amount of papain added in step 22) is 1500-1800U / L, and the amount of bromelain added is 500-1000U / L.

7. A method for purifying β-1,3-glucan from a fermentation broth of Saccharomyces cerevisiae cells according to claim 5, characterized in that , the alkali in step 23) is sodium hydroxide.

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