A low-endotoxin yeast extract, its preparation method and application

Abstract

The present invention relates to a low endotoxin yeast extract, a preparation method thereof and an application thereof. Calculated by dry basis weight percentage, the total nitrogen content in the low endotoxin yeast extract is 10-13%, the content of amino acid nitrogen is 4-6%, and the endotoxin content is ≤20 EU / g. The low endotoxin yeast extract prepared by the present invention is applied to cell culture, will not bring toxicity to cells, is beneficial to the purification and quality control of target products, can replace serum, and effectively improves the cell proliferation rate.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and particularly relates to a low-endotoxin yeast extract, a preparation method thereof, and an application thereof. Background Art

[0002] Yeast extract is a product in the form of powder, paste or liquid obtained by using fresh yeast as a raw material, inducing various enzyme systems in the yeast itself by means of modern biotechnology or by changing the medium environment, adding (or not adding) certain exogenous enzymes, and promoting the degradation of intracellular proteins and nucleic acid substances under suitable conditions, and then through some refining processes. It is rich in proteins, amino acids, peptides, nucleotides, B vitamins, and trace elements. Its main function is to supplement nitrogen sources and provide various vitamins, amino acids and growth factors for microbial growth. It is widely used in the fermentation medium industry and is a culture medium raw material with strong growth-promoting ability.

[0003] Endotoxin is a component in the cell membrane of Gram-negative bacteria. Its main component is lipopolysaccharide, also known as LPS, and its main toxic component is lipid A. The outer membrane of the cell wall of Gram-negative bacteria is entirely composed of endotoxin molecules. An Escherichia coli cell contains about 2 million LPS molecules. Bacteria secrete endotoxin into the surrounding environment when they are active or die in large numbers. The toxic reactions caused by endotoxin mainly include symptoms such as fever, microcirculation disorders, disseminated intravascular coagulation, etc. In severe cases, it will cause endotoxin shock.

[0004] The endotoxin in yeast extract is usually released into the product due to the death of Gram-negative bacteria caused by factors such as high temperature. Yeast extract is often used as an additive in the culture of animal cells. In addition to meeting the needs of cell growth, animal cell culture media also require that the endotoxin content of various raw materials in the culture media must be controlled within the specified range.

[0005] Endotoxin has good thermal stability and chemical stability. Currently, the methods for removing endotoxin mainly include chemical destruction methods based on strong acids, alkalis, and oxidants, dry heat methods based on high temperature, and ion exchange methods and molecular sieve methods based on various separation devices. Currently, various methods have high requirements for equipment and high costs. Currently, there is no method for removing or reducing endotoxin during the production process of yeast extract. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that in the prior art, the endotoxin content in yeast extract is relatively high, which is not conducive to cell growth and is also not conducive to the purification of downstream products, bringing safety risks.

[0007] In view of the deficiencies in the prior art, one of the objectives of the present invention is to provide a low endotoxin yeast extract. Another objective of the present invention is to provide a preparation method for the above-mentioned low endotoxin yeast extract. A third objective of the present invention is to provide the application of the above-mentioned low endotoxin yeast extract in biomedicine and cell culture.

[0008] Technical solution of the present invention:

[0009] The present invention provides a low endotoxin yeast extract. Based on the dry weight percentage, the total nitrogen content in the low endotoxin yeast extract is 10-13%, the content of amino acid nitrogen is ≥5%, and the endotoxin content is ≤20 EU / g.

[0010] Preferably, the endotoxin content in the above-mentioned low endotoxin yeast extract is ≤10 EU / g.

[0011] Preferably, the pH value of the aqueous solution of the above-mentioned low endotoxin yeast extract with a weight percentage concentration of 2-5% is 4.5-7.0.

[0012] Preferably, the above-mentioned low endotoxin yeast extract is in liquid or powder form.

[0013] The present invention also provides a preparation method for the above-mentioned low endotoxin yeast extract, which includes the following steps:

[0014] (1) Autolysis and enzymatic hydrolysis: Autolyze the yeast slurry at 40-60°C and pH 4.5-6.0, and then add protease for enzymatic hydrolysis;

[0015] (2) Separation and purification: Subject the above-mentioned autolyzed and enzymatically hydrolyzed solution to centrifugal separation, membrane separation, and evaporation processes in sequence to obtain a liquid low endotoxin yeast extract. Among them, the membrane separation is one or more of microfiltration, ultrafiltration, or nanofiltration. The pore size of the microfiltration membrane is 0.1-0.2 μm, the pore size of the ultrafiltration membrane is 10-90 nm, and the pore size of the nanofiltration membrane is 0.1-1 nm.

[0016] Preferably, in the above-mentioned preparation method, the preparation method of the yeast slurry is as follows:

[0017] A. Prepare a yeast seed solution with yeast;

[0018] B. Prepare a culture medium, adjust the pH of the culture medium to 4.5-5.0, add the yeast seed solution, and ferment at 30-35°C to obtain a yeast fermentation broth;

[0019] C. Wash the yeast fermentation broth with water and perform centrifugal separation to obtain yeast milk;

[0020] D. Add water to the yeast milk to prepare a slurry with a dry matter weight content of 10-15%.

[0021] Preferably, the yeast described in step A is one of Saccharomyces cerevisiae, Pichia pastoris or Candida yeast.

[0022] Preferably, in the above method for preparing low endotoxin yeast extract, based on the dry matter weight of the yeast slurry, the addition amount of the protease is 0.02 - 0.08%.

[0023] Preferably, in the above method for preparing low endotoxin yeast extract, in step (2), the centrifugal separation speed is 4000 - 8000 rpm and the time is 10 - 30 min. Through centrifugal separation, the insoluble part and the soluble part in the autolyzed enzymolysis product are separated, and the supernatant is collected for subsequent separation operations.

[0024] Preferably, in the above method for preparing low endotoxin yeast extract, in step (1), the autolysis process is carried out for 6 - 10 h, and preferably the enzymolysis is carried out for 10 - 14 h.

[0025] Preferably, in the above method for preparing low endotoxin yeast extract, the process water used in the preparation process is all ultrapure water or water for injection.

[0026] Specifically, the process water used in the preparation process includes the water used for preparing the culture medium in the yeast slurry preparation process, the water for washing the yeast fermentation broth, the water for adjusting the yeast slurry concentration, and the water for washing in the centrifugal separation.

[0027] Preferably, in the above method for preparing low endotoxin yeast extract, the evaporation process in step (2) is a multi-effect evaporation process. Preferably, the weight percentage concentration of the concentrated liquid obtained by the evaporation process is 20 - 60%, and further preferably, the evaporation temperature is 65 - 80 °C.

[0028] Preferably, the above preparation method further includes a drying process to obtain a powdery low endotoxin yeast extract.

[0029] Preferably, the drying is spray drying. Preferably, the inlet air temperature for drying is 200 - 300 °C and the outlet air temperature is 100 - 150 °C.

[0030] The present invention also provides the application of the above low endotoxin yeast extract or the low endotoxin yeast extract prepared by the above method for preparing low endotoxin yeast extract in biomedicine and cell culture.

[0031] The beneficial effects of the present invention:

[0032] The present invention uses yeast autolysis, enzymatic hydrolysis, separation and purification to obtain a low endotoxin yeast extract, with an endotoxin content not exceeding 20 EU / g. High-temperature drying can further improve the product quality and control the endotoxin content in the final product ≤ 10 EU / g. The low endotoxin yeast extract prepared by the present invention is applied to cell culture, will not bring toxicity to cells, is conducive to the purification and quality control of the target product, can replace serum, and effectively improve the cell proliferation rate. Detailed implementation mode

[0033] One of the purposes of the present invention is to provide a low endotoxin yeast extract.

[0034] Specifically, a low endotoxin yeast extract, calculated by dry basis weight percentage, has a total nitrogen content of 10-13% in the low endotoxin yeast extract, an amino acid nitrogen content ≥ 5%, and an endotoxin content ≤ 20 EU / g.

[0035] Preferably, the endotoxin content in the above low endotoxin yeast extract is ≤ 10 EU / g.

[0036] Preferably, the pH of the aqueous solution of the above low endotoxin yeast extract with a weight percentage concentration of 2-5% is 4.5-7.0.

[0037] Preferably, the above low endotoxin yeast extract is in liquid or powder form.

[0038] Another purpose of the present invention is to provide a preparation method of the above low endotoxin yeast extract, including the following steps:

[0039] (1) Autolysis and enzymatic hydrolysis: Autolyze the yeast slurry at 40-60 °C and pH 4.5-6.0, and then add protease for enzymatic hydrolysis;

[0040] (2) Separation and purification: Subject the above autolyzed and enzymatically hydrolyzed solution to centrifugal separation, evaporation process and membrane separation in sequence to obtain a liquid low endotoxin yeast extract. Among them, the membrane separation is one or more of microfiltration, ultrafiltration or nanofiltration. The pore size of the microfiltration membrane is 0.1-0.2 μm, the pore size of the ultrafiltration membrane is 10-90 nm, and the pore size of the nanofiltration membrane is 0.1-1 nm. By using the retention effect of the membrane on molecules, endotoxin in the yeast extract can be removed.

[0041] Preferably, in the above preparation method, the preparation method of the yeast slurry is as follows:

[0042] A. Prepare a yeast seed solution with yeast;

[0043] B. Prepare a culture medium, adjust the pH of the culture medium to 4.5-5.0, add the yeast seed solution, and ferment at 30-35 °C to obtain a yeast fermentation broth;

[0044] C. washing the yeast fermentation liquid with water and centrifuging to obtain yeast milk;

[0045] D. Add water to the yeast milk to prepare a slurry with a dry matter weight content of 10-15%.

[0046] Preferably, the yeast in step A is one of Saccharomyces cerevisiae, Pichia pastoris or Candida.

[0047] Preferably, in the method for preparing the low-endotoxin yeast extract, the amount of the protease added is 0.02-0.08% based on the weight of the dry matter of the yeast slurry.

[0048] Preferably, in the method for preparing low-endotoxin yeast extract, the centrifugal separation speed in step (2) is 4000-8000 rpm and the time is 10-30 min. By centrifugal separation, the insoluble part and the soluble part of the autolysis product are separated, and the supernatant is collected for subsequent separation operations.

[0049] Preferably, in the method for preparing the low-endotoxin yeast extract, in step (1), the autolysis process is carried out for 6-10 hours, and preferably the enzymatic hydrolysis is carried out for 10-14 hours.

[0050] Preferably, in the method for preparing low-endotoxin yeast extract, the process water used in the preparation process is ultrapure water or water for injection. Since the endotoxin content in ultrapure water or water for injection is very low, the effect of process water on the endotoxin content is reduced, further ensuring that the endotoxin content in the final product is ≤20EU / g.

[0051] Specifically, the process water used in the preparation process includes water used to prepare the culture medium during the yeast slurry preparation process, water used to wash the yeast fermentation liquid, water used to adjust the yeast slurry concentration, and water used for centrifugal separation and washing.

[0052] Preferably, in the method for preparing low-endotoxin yeast extract, the evaporation process in step (2) is a multiple-effect evaporation process. Preferably, the concentration of the concentrated solution obtained by the evaporation process is 20-60%. More preferably, the evaporation temperature is 65-80°C. On the one hand, after evaporation and concentration, the material concentration is increased, which can effectively reduce the microbial reproduction rate, thereby reducing the production of endotoxins; on the other hand, in the production process of yeast extract, each process link has the risk of microbial contamination. Since membrane separation can remove endotoxins in the product, placing membrane separation after the evaporation process can reduce the impact of microbial contamination in the operation before membrane separation on the endotoxin content of the final product, and is also conducive to reducing the endotoxin content in the final product.

[0053] Preferably, the above preparation method further comprises a drying process to obtain a powdery low-endotoxin yeast extract.

[0054] Preferably, the drying is spray drying. Preferably, the inlet air temperature for drying is 200 - 300 °C, and the outlet air temperature is 100 - 150 °C. Yeast extract is very rich in nutrients and is very easy to breed microorganisms. During the preparation of the extract, due to production fluctuations, the endotoxin content in the final product may fluctuate. If the inlet air temperature for drying is 200 - 300 °C and the outlet air temperature is 100 - 150 °C, the product quality can be further improved, and the endotoxin content in the final product can be controlled to ≤ 10 EU / g.

[0055] The third object of the present invention is to provide the application of the above-mentioned low-endotoxin yeast extract or the low-endotoxin yeast extract prepared by the above-mentioned method for preparing low-endotoxin yeast extract in biomedicine and cell culture.

[0056] Strain preservation information:

[0057] The Saccharomyces cerevisiae FX-2 used in the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on August 1, 2016, with the deposit number CCTCC NO: M2016418. This strain has been recorded in the patent publication text with the publication number CN108220175A.

[0058] The Wickerhamomyces anomalus C1.7 used in the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on December 11, 2017, with the deposit number CCTCC NO: M2017782. This strain has been recorded in the patent publication text with the publication number CN110959853A.

[0059] The Cyberlindnera fabianii C1.8 used in the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on December 11, 2017, with the deposit number CCTCC NO: M2017780. This strain has been recorded in the patent publication text with the publication number CN110959853A.

[0060] After testing, the physical and chemical indexes of the endotoxin yeast extract prepared by the preparation method of the present invention are as shown in Table 1:

[0061] Table 1 Physical and chemical indexes of endotoxin yeast extract

[0062]

[0063] The present invention has undergone strict preliminary tests, proving that this method is feasible.

[0064] It can be seen from this that the preparation method described in the present invention can effectively reduce the endotoxin level in yeast extract. The endotoxin content in the prepared low-endotoxin yeast extract product is ≤20 EU / g, which can be applied to cell culture to replace serum and improve the production efficiency in the field of cell culture.

[0065] The present invention will be specifically described below through specific examples and experimental examples.

[0066] The detection methods for various indicators of yeast extract in the following examples and comparative examples are as follows:

[0067] (1) Determination of moisture

[0068] Adopt the method in 6.2 of the national standard GB / T 23530-2009. Take a certain mass of the sample, dry it at 103 °C for 4 hours until constant weight, and weigh and calculate the moisture content.

[0069] (2) Determination of total nitrogen

[0070] Adopt the Kjeldahl method in 6.4 of the national standard GB / T 23530-2009: Take the sample (equivalent to 30-440 mg of total nitrogen), under the action of 5 g of mixed catalyst a (potassium sulfate and sewage copper sulfate 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), add 20 mL of concentrated sulfuric acid for digestion; then distill, absorb the product ammonia with boric acid; then titrate with 0.1 mol / L hydrochloric acid, read the data, and calculate the total nitrogen content.

[0071] (3) Determination of amino acid nitrogen

[0072] Adopt the detection method of amino acid nitrogen described in 6.5 of the national standard GB / T 23530-2009: Take 5 g of the sample, dilute it and titrate it with 0.5 mol / L sodium hydroxide solution until the pH is 8.2 and keep it for 1 min. Slowly add 10 mL of 36% formaldehyde solution to react with the non-dissociated amino groups in neutral amino acids to form mono-hydroxymethyl and di-hydroxymethyl derivatives. This reaction proceeds quantitatively. The hydrogen ions released at this time are titrated with the above sodium hydroxide, and the content of amino acid nitrogen is calculated according to the consumption of the alkali solution.

[0073] (4) Determination of pH

[0074] Adopt the pH detection method described in 7.2.1 of the national standard GB / T 35536-2017: Prepare an aqueous solution with a certain weight percentage concentration, and use a glass electrode to measure the pH value after sterilization. The difference between the two measured values of the same sample should not exceed 0.04.

[0075] (5) Determination of endotoxin

[0076] Weigh about 0.01 - 0.05 g of the sample into a pyrogen-free test tube, add an appropriate amount of water for bacterial endotoxin test to dissolve it, and make the concentration exactly 10 mg / ml. Then dilute it with water for bacterial endotoxin test to the required detection multiple respectively, and the maximum dilution multiple shall not exceed 20 times. Use pH test paper to detect the acidity and alkalinity. If it is slightly acidic, it can be adjusted with 0.1 N sodium hydroxide. It is recommended to pipette 5 μl each time for addition, in small amounts and multiple times, so as to avoid deviation during adjustment. The amount of reagent added here can be ignored. Take pyrogen-free test tubes, add 100 μl of water for bacterial endotoxin test, endotoxin standard solution, and diluted sample solution respectively, quickly add 100 μl of limulus reagent solution; gently shake the test tube rack to mix evenly, cover all test tubes with a whole piece of pyrogen-free tin foil paper, and incubate in a 37 °C water bath for T1 minutes; after incubation, quickly add 100 μl of chromogenic substrate solution; gently shake the test tube rack to mix evenly, cover all test tubes with a whole piece of pyrogen-free tin foil paper, and incubate in a 37 °C water bath for T2 minutes; after incubation, add 500 μl of diazotization reagent 1 solution, mix gently; add 500 μl of diazotization reagent 2 solution, mix gently; add 500 μl of diazotization reagent 3 solution, mix gently, let it stand for 5 minutes, and read the absorbance value at a wavelength of 545 nm.

[0077] Note: The times of T1 and T2 can be found in the factory inspection report of this batch of limulus reagents.

[0078] The sources of raw materials and equipment used in the examples, comparative examples and experimental examples of the present invention are shown in Table 2.

[0079] Table 2 Sources of Raw Materials and Equipment Used in Examples and Comparative Examples of the Present Invention

[0080]

[0081]

[0082] Example 1

[0083] The preparation method of low-endotoxin yeast extract is as follows:

[0084] 1. Preparation of yeast slurry

[0085] Take 5 g of Saccharomyces cerevisiae, add it to 50 mL of 2 wt% glucose solution, and activate it at 28 °C for 30 min to obtain a Saccharomyces cerevisiae seed solution. Then, add 5 kg of molasses, 0.5 kg of yeast extract powder, 2 kg of ammonium sulfate, 0.5 kg of magnesium sulfate, 0.3 kg of potassium dihydrogen phosphate, 0.1 kg of zinc sulfate, and 91.6 kg of ultrapure water to the fermenter, stir and dissolve, adjust the pH to 4.5, prepare the medium, and then add 1500 ml of Saccharomyces cerevisiae seed solution to the fermenter and ferment at 30 °C for 18 h to obtain a yeast fermentation broth. Then, wash the yeast fermentation broth with deionized water, centrifuge and separate, repeat 2 times to obtain yeast cream. Add deionized water to the yeast cream to prepare a yeast slurry with a dry matter weight content of 15%.

[0086] 2. Autolysis and enzymatic hydrolysis

[0087] Adjust the pH of the yeast slurry prepared in step 1 to 5.0, raise the temperature to 40 °C, autolyze for 10 h, and then add 0.02% (calculated based on the dry matter weight of the yeast slurry) of protease and enzymatically hydrolyze for 14 h to obtain an enzymatic hydrolysate.

[0088] 3. Separation and purification

[0089] (1) Centrifugal separation: Centrifuge the enzymatic hydrolysate obtained in step 2 at 8000 rpm for 10 min, collect the supernatant, wash the precipitate with ultrapure water, then centrifuge again at 8000 rpm for 10 min, collect the supernatant, and mix the supernatants obtained twice.

[0090] (2) Evaporation and concentration: Concentrate the supernatant obtained in step (1) to a dry matter weight percentage concentration of 20% using a multi-effect evaporation process, and the evaporation temperature is 65 °C.

[0091] (3) Microfiltration membrane separation: Filter the concentrated solution obtained in step (2) using a microfiltration membrane with a pore size of 0.2 μm to obtain a filtrate, where: the flow rate during microfiltration is 4 - 5 L / h, and the operating temperature is 50 - 65 °C.

[0092] (4) Drying: Dry the filtrate obtained in step (3) in a spray dryer, with an inlet air temperature of 200 °C and an outlet air temperature of 100 °C, to obtain a powdery low endotoxin yeast extract with a moisture content of less than 6%.

[0093] Example 2

[0094] A method for preparing a low endotoxin yeast extract, the steps are as follows:

[0095] 1. Preparation of yeast slurry

[0096] Use Pichia pastoris to replace Saccharomyces cerevisiae, and the others are the same as in Example 1.

[0097] 2. Autolysis and enzymatic hydrolysis

[0098] Adjust the pH of the yeast slurry prepared in Step 1 to 5.5, heat it up to 50 °C, autolyze for 8 h, then add 0.04% (based on the dry matter weight of the yeast slurry) protease, and enzymatically hydrolyze for 12 h to obtain an enzymolysis solution.

[0099] 2. Separation and purification

[0100] (1) Centrifugal separation: Centrifuge the enzymolysis solution obtained in Step 2 at 6000 rpm for 20 min, collect the supernatant, wash the precipitate with injection water, then centrifuge again at 6000 rpm for 20 min, collect the supernatant, and mix the supernatants obtained twice.

[0101] (2) Evaporation and concentration: Concentrate the supernatant obtained in Step (1) to a dry matter weight percentage concentration of 30% using a multi-effect evaporation process, and the evaporation temperature is 75 °C.

[0102] (3) Microfiltration membrane separation: Filter the concentrated solution obtained in Step (1) with a microfiltration membrane with a pore size of 0.1 μm to obtain a filtrate. The flow rate during microfiltration is 4 - 5 L / h, and the operating temperature is 50 - 65 °C.

[0103] (4) Drying: Dry the filtrate obtained in Step (3) in a spray dryer, with an inlet air temperature of 300 °C and an outlet air temperature of 150 °C, to obtain a powdery low endotoxin yeast extract with a moisture content of less than 6%.

[0104] Example 3

[0105] A method for preparing a low endotoxin yeast extract, the steps are as follows:

[0106] 1. Preparation of yeast slurry

[0107] Use Candida instead of Saccharomyces cerevisiae, and the others are the same as in Example 1.

[0108] 2. Autolysis and enzymatic hydrolysis

[0109] Adjust the pH of the yeast slurry prepared in Step 1 to 6.0, heat it up to 60 °C, autolyze for 6 h, then add 0.08% (based on the dry matter weight of the yeast slurry) protease, and enzymatically hydrolyze for 10 h to obtain an enzymolysis solution.

[0110] 3. Separation and purification

[0111] (1) Centrifugal separation: Centrifuge the enzymolysis solution obtained in Step 2 at 4000 rpm for 30 min, collect the supernatant, wash the precipitate with ultrapure water, then centrifuge again at 4000 rpm for 30 min, collect the supernatant, and mix the supernatants obtained twice.

[0112] (2) Evaporation and concentration: The supernatant obtained in step (1) is concentrated to a dry matter weight percentage concentration of 40% by a multi-effect evaporation process, and the evaporation temperature is 80°C.

[0113] (3) Membrane separation: The concentrated liquid obtained in step (2) is filtered successively through a microfiltration membrane with a pore size of 0.2 μm and an ultrafiltration membrane with a pore size of 90 nm to obtain a filtrate. Among them, the flow rate during microfiltration is 4 - 5 L / h, and the operating temperature is 50 - 65°C; the flow rate during ultrafiltration is 4 - 5 L / h, and the operating temperature is 50 - 65°C.

[0114] (4) Drying: The filtrate obtained in step (3) is dried in a spray dryer with an inlet air temperature of 250°C and an outlet air temperature of 125°C to obtain a powdery low endotoxin yeast extract with a moisture content of less than 6%.

[0115] Example 4

[0116] A method for preparing a low endotoxin yeast extract, the steps are as follows:

[0117] 1. Preparation of yeast slurry

[0118] Same as in Example 1.

[0119] 2. Autolysis and enzymolysis

[0120] Adjust the pH of the yeast slurry prepared in step 1 to 6.0, heat it to 40°C, autolyze for 6 h, and then add 0.05% (calculated based on the dry matter weight of the yeast slurry) protease and enzymolyze for 10 h to obtain an enzymolyzed solution.

[0121] 3. Separation and purification

[0122] (1) Centrifugal separation: The enzymolyzed solution obtained in step 2 is centrifuged at 5000 rpm for 30 min, the supernatant is collected, and the precipitate is washed with injection water, and then centrifuged again at 5000 rpm for 30 min, the supernatant is collected, and the supernatants obtained twice are mixed.

[0123] (2) Evaporation and concentration: The supernatant obtained in step (1) is concentrated to a dry matter weight percentage concentration of 50% by a multi-effect evaporation process, and the evaporation temperature is 65°C.

[0124] (3) Ultrafiltration membrane separation: The concentrated liquid obtained in step (2) is filtered through an ultrafiltration membrane with a pore size of 10 nm to obtain a filtrate, and the flow rate during ultrafiltration is 4 - 5 L / h, and the operating temperature is 50 - 65°C.

[0125] (4) Drying: The filtrate obtained in step (3) is dried in a spray dryer with an inlet air temperature of 250°C and an outlet air temperature of 125°C to obtain a powdery low endotoxin yeast extract with a moisture content of less than 6%.

[0126] Example 5

[0127] A preparation method of low endotoxin yeast extract comprises the following steps:

[0128] 1. Preparation of yeast slurry

[0129] The same as in Example 1.

[0130] 2. Autolysis and enzymolysis

[0131] Adjust the pH of the yeast slurry prepared in step 1 to 6.0, heat it up to 40 °C, autolyze for 6 h, then add 0.05% (calculated based on the dry matter weight of the yeast slurry) of protease and enzymolyze for 10 h to obtain an enzymolyzed solution.

[0132] 3. Separation and purification

[0133] (1) Centrifugal separation: Centrifuge the enzymolyzed solution obtained in step 2 at 5000 rpm for 30 min, collect the supernatant, wash the precipitate with injection water, then centrifuge again at 5000 rpm for 30 min, collect the supernatant, and mix the supernatants obtained twice.

[0134] (2) Evaporation and concentration: Concentrate the supernatant obtained in step (1) to a dry matter weight percentage concentration of 50% by a multi-effect evaporation process, and the evaporation temperature is 65 °C.

[0135] (3) Ultrafiltration membrane separation: Filter the concentrated solution obtained in step (2) with an ultrafiltration membrane with a pore size of 10 nm to obtain a filtrate. The flow rate during ultrafiltration is 4 - 5 L / h, and the operating temperature is 50 - 65 °C.

[0136] Example 6

[0137] A preparation method of low endotoxin yeast extract comprises the following steps:

[0138] 1. Preparation of yeast slurry

[0139] The same as in Example 1.

[0140] 2. Autolysis and enzymolysis

[0141] Adjust the pH of the yeast slurry prepared in step 1 to 6.0, heat it up to 40 °C, autolyze for 6 h, then add 0.05% (calculated based on the dry matter weight of the yeast slurry) of protease and enzymolyze for 10 h to obtain an enzymolyzed solution.

[0142] 3. Separation and purification

[0143] (1) Centrifugal separation: Centrifuge the enzymolyzed solution obtained in step 2 at 5000 rpm for 30 min, collect the supernatant, wash the precipitate with ultrapure water, then centrifuge again at 5000 rpm for 30 min, collect the supernatant, and mix the supernatants obtained twice.

[0144] (2) Evaporation and concentration: The supernatant obtained in step (1) is concentrated to a dry matter weight percentage concentration of 50% by a multi-effect evaporation process, and the evaporation temperature is 65°C.

[0145] (3) Membrane separation: The concentrated liquid obtained in step (2) is filtered through a nanofiltration membrane with a pore size of 1 nm to obtain a filtrate. During nanofiltration, the flow rate is 4 - 5 L / h, and the operating temperature is 50 - 65°C.

[0146] (4) Drying: The filtrate obtained in step (3) is dried in a spray dryer with an inlet air temperature of 250°C and an outlet air temperature of 125°C to obtain a powdery low endotoxin yeast extract with a moisture content of less than 6%.

[0147] Example 7

[0148] A method for preparing a low endotoxin yeast extract, the steps are as follows:

[0149] 1. Preparation of yeast slurry

[0150] Same as in Example 1.

[0151] 2. Autolysis and enzymolysis

[0152] Adjust the pH of the yeast slurry prepared in step 1 to 6.0, heat it to 40°C, autolyze for 6 h, and then add 0.05% (calculated based on the dry matter weight of the yeast slurry) protease and enzymolyze for 10 h to obtain an enzymolyzed solution.

[0153] 3. Separation and purification

[0154] (1) Centrifugal separation: The enzymolyzed solution obtained in step 2 is centrifuged at 5000 rpm for 30 min, the supernatant is collected, and the precipitate is washed with ultrapure water, then centrifuged again at 5000 rpm for 30 min, the supernatant is collected, and the supernatants obtained twice are mixed.

[0155] (2) Evaporation and concentration: The supernatant obtained in step (1) is concentrated to a dry matter weight percentage concentration of 50% by a multi-effect evaporation process, and the evaporation temperature is 65°C.

[0156] (3) Membrane separation: The concentrated liquid obtained in step (2) is filtered through a ultrafiltration membrane with a pore size of 10 nm to obtain a filtrate. During ultrafiltration, the flow rate is 4 - 5 L / h, and the operating temperature is 50 - 65°C.

[0157] (4) Drying: The filtrate obtained in step (3) is dried in a spray dryer with an inlet air temperature of 130°C and an outlet air temperature of 80°C to obtain a powdery low endotoxin yeast extract with a moisture content of less than 6%.

[0158] Comparative Example 1

[0159] The preparation method of conventional yeast extract is as follows:

[0160] 1. Preparation of yeast slurry

[0161] Take 5 g of Saccharomyces cerevisiae, add it to 50 mL of 2 wt% glucose solution, activate it at 28 °C for 30 min to obtain a Saccharomyces cerevisiae seed solution. Then add 5 kg of molasses, 0.5 kg of yeast extract powder, 2 kg of ammonium sulfate, 0.5 kg of magnesium sulfate, 0.3 kg of potassium dihydrogen phosphate, 0.1 kg of zinc sulfate and 91.6 kg of deionized water to the fermenter, stir and dissolve, adjust the pH to 4.5 to prepare a culture medium, and then add 1500 ml of Saccharomyces cerevisiae seed solution to the fermenter, ferment at 30 °C for 18 h to obtain a yeast fermentation broth. Then wash the yeast fermentation broth with deionized water, centrifuge and separate, repeat 2 times to obtain yeast cream. Add deionized water to the yeast cream to prepare a yeast slurry with a dry matter weight content of 15%.

[0162] 2. Autolysis and enzymatic hydrolysis

[0163] Adjust the pH of the yeast slurry prepared in step 1 to 6.0, raise the temperature to 40 °C, autolyze for 6 h, and then add 0.05% (calculated based on the dry matter weight of the yeast slurry) protease and enzymatically hydrolyze for 10 h to obtain an enzymatic hydrolysate.

[0164] 3. Separation and purification

[0165] (1) Centrifugal separation: Centrifuge the enzymatic hydrolysate obtained in step 2 at 5000 rpm for 30 min, collect the supernatant, wash the precipitate with deionized water, and then centrifuge again at 5000 rpm for 30 min, collect the supernatant, and mix the supernatants obtained twice.

[0166] (2) Filter the supernatant in step (1) with a membrane filter with an average pore size of 10 μm to obtain a filtrate.

[0167] (3) Evaporation and concentration: Concentrate the filtrate obtained in step (2) to a dry matter weight percentage concentration of 50% by a multi-effect evaporation process, and the evaporation temperature is 65 °C.

[0168] (4) Drying: Dry the concentrated solution obtained in step (3) in a spray dryer, with an inlet air temperature of 130 °C and an outlet air temperature of 80 °C, to obtain a powdery yeast extract with a water content of less than 6%.

[0169] Detect the physical and chemical indexes of Examples 1-7 and Comparative Example 1. Among them, total nitrogen, amino acid nitrogen and endotoxin are all calculated based on the dry matter weight, and the results are shown in Table 3.

[0170] Table 3 Detection results of physical and chemical indexes of Examples 1-7 and Comparative Example 1

[0171]

[0172]

[0173] Experimental Example 1

[0174] Cell culture tests were performed on the low endotoxin yeast extracts prepared in Examples 1-7, the conventional yeast extract in Comparative Example 1, and serum to test the cell proliferation rate.

[0175] The method for testing the cell proliferation rate is as follows:

[0176] Take CHO-K1 suspension cells in the exponential growth phase, centrifuge at 1000 r / min for 5 min, discard the supernatant, and resuspend with fresh commercial CHO medium. Inoculate into a 12-well plate at a live cell density of 8.0×10 5 cells / ml, inoculate 3 ml of commercial CHO cell medium into each well, culture in 5% CO2 for 6 d, take 100 μL every 24 h and mix it evenly with trypan blue at a ratio of 1:1, and count the cells and determine the cell viability using a cell counter.

[0177] Using no hydrolyzate as a blank control, the addition concentration of the yeast extracts in each example and comparative example was 1 g / L, and the addition amount of serum in the serum group was 10%. Three parallels were set for each group of experiments, and Table 4 shows the average results of the three parallels.

[0178] The experimental results are shown in Table 4:

[0179] Table 4 Cell proliferation test results of Examples 1-7, Comparative Example 1 and serum, unit: Cells / ml

[0180]

[0181]

[0182] As can be seen from Table 3, the endotoxin content of the yeast extracts prepared in Examples 1-7 of the present invention is ≤20 EU / g. By using a higher drying temperature, the endotoxin content can reach below 10 EU / g, which is far lower than the endotoxin level of the yeast extract in Comparative Example 1. As can be seen from Table 4, after 4 days of cell proliferation culture, the cell proliferation rate of the present invention is significantly higher than that of the blank and Comparative Example 1, and is comparable to the serum level, indicating that the present invention applied to cell culture will not bring toxicity to cells, is beneficial to the purification and quality control of the target product, can replace serum, and effectively improve the cell proliferation rate.

[0183] The above are only the preferred embodiments of the implementation of the present invention, and do not impose any form of limitation on the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a low endotoxin yeast extract, characterized in that, It includes the following steps: (1) Autolysis and enzymatic hydrolysis: Autolyze the yeast pulp at 40 - 60 °C and pH 4.5 - 6.0 for 6 - 10 h, and then add protease for enzymatic hydrolysis for 10 - 14 h; among them, the yeast for preparing the yeast pulp is from one of Saccharomyces cerevisiae, Pichia pastoris or Candida sp., and based on the dry matter weight of the yeast pulp, the addition amount of the protease is 0.02 - 0.08%; the Saccharomyces cerevisiae is Saccharomyces cerevisiae FX - 2, and the preservation number is CCTCC NO: M2016418, the Pichia pastoris is Pichia pastoris C1.8, and the preservation number is CCTCC NO: M2017780, the Candida sp. is Candida sp. C1.7, and the preservation number is CCTCC NO: M2017782; (2) Separation and purification: Subject the above autolyzed and enzymatically hydrolyzed solution to centrifugal separation, evaporation process, membrane separation and drying in sequence to obtain powdery low - endotoxin yeast extract. Among them, the membrane separation is one or more of microfiltration, ultrafiltration or nanofiltration. The pore size of the microfiltration membrane is 0.1 - 0.2 μm, the pore size of the ultrafiltration membrane is 10 - 90 nm, and the pore size of the nanofiltration membrane is 0.1 - 1 nm; the drying is spray drying, the inlet air temperature for drying is 200 - 300 °C, and the outlet air temperature is 100 - 150 °C; Among them, based on the dry - basis weight percentage, the total nitrogen content in the low - endotoxin yeast extract is 10 - 13%, the content of amino - acid - state nitrogen is 4 - 6%, and the endotoxin content ≤ 10 EU / g.

2. The preparation method of the low endotoxin yeast extract according to claim 1, characterized in that, The pH of the aqueous solution of the low - endotoxin yeast extract with a weight - percentage concentration of 2 - 5% is 4.5 - 7.

0.

3. The preparation method according to claim 1, wherein The rotation speed of the centrifugal separation in step (2) is 4000 - 8000 rpm, and the time is 10 - 30 min.

4. The preparation method according to claim 2, characterized in that, The rotation speed of the centrifugal separation in step (2) is 4000 - 8000 rpm, and the time is 10 - 30 min.

5. According to the preparation method described in any one of claims 1-4, it is characterized in that, The evaporation process is a multi - effect evaporation process.

6. The preparation method according to claim 5, wherein The weight - percentage concentration of the concentrated solution obtained by the evaporation process is 20 - 60%.

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