CN105695350B

CN105695350B - Saccharomyces cerevisiae and method for producing glucosamine by using saccharomyces cerevisiae

Info

Publication number: CN105695350B
Application number: CN201610168668.6A
Authority: CN
Inventors: 张春颖
Original assignee: Tibet Tianhong Technology Co ltd
Current assignee: Tibet Tianhong Technology Co ltd
Priority date: 2016-03-23
Filing date: 2016-03-23
Publication date: 2020-02-07
Anticipated expiration: 2036-03-23
Also published as: CN105695350A

Abstract

The invention discloses a saccharomyces cerevisiae and a method for producing glucosamine by the saccharomyces cerevisiae, wherein the saccharomyces cerevisiae is preserved in the China general microbiological culture Collection center. Seed culture: inoculating the saccharomyces cerevisiae into a slant culture medium containing kanamycin sulfate, culturing for 24-28 h, inoculating into a seed culture medium, and culturing to OD₆₀₀When the value is 2-3, obtaining a seed culture solution; seed fermentation: inoculating the planting culture solution to a fermentation culture medium, and fermenting for 18-20 h to obtain a fermentation liquid; the volume of the seed culture solution inoculated to the fermentation culture medium is 1 to 2 percent of the volume of the fermentation culture medium; purifying and crystallizing the fermentation liquor to obtain the glucosamine. The invention optimizes the fermentation process of the mutagenized engineering strain, and determines that the yield of the glucosamine is obviously improved by implementing a lactose feeding strategy and a staged dissolved oxygen control strategy in the fermentation process.

Description

Saccharomyces cerevisiae and method for producing glucosamine by using saccharomyces cerevisiae

Technical Field

The invention relates to saccharomyces cerevisiae and a method for producing glucosamine by using the saccharomyces cerevisiae.

Background

Glucosamine (glucamine, or 2-amino-2-deoxy-D-glucose), the English abbreviation of which is GlcN, is a substituted compound in which the hydroxyl group at C at position 2 of glucose is substituted with an amino group, and the first amino monosaccharide to be confirmed is Glucosamine, which is a very important functional monosaccharide. The molecular formula of GlcN is C₆H₁₃O₅N, molecular weight 179.17 g/mL. GlcN is found in many organisms, including bacteria, filamentous fungi, yeast, and even in plants and animals. Glucosamine is an important component of chitin, chitosan, and glycoprotein. Studies have also shown that glucosamine plays an important role in proteoglycans, all of which can be obtained from glucosamine by a certain amination. In addition, as a physiologically essential substance, it is also present in human connective tissues and is an important component necessary for the synthesis of proteoglycan in the matrix of human cartilage. The glucosamine has wide application and wide market prospect.

In 1876, glucosamine was first isolated from the hydrolysate of chitin, and in 1939, Walter Howski determined the spatial structure of glucosamine, from which glucosamine was better known and studied. At present, the united states is the largest country of consumption of glucosamine, and along with the improvement of income level and health care consciousness in china, the consumption of glucosamine is also in a trend of rapid increase year by year.

The application of glucosamine in the pharmaceutical industry is as follows:

glucosamine sulfate is a raw material drug for treating rheumatoid arthritis, and in recent years, glucosamine sulfate has various physiological functions of absorbing free radicals generated in a body, resisting aging, promoting weight loss, inhibiting bacteria, regulating endocrine of a human body and the like, so that glucosamine sulfate is widely used for production of food additives and health-care food. GlcN has a special therapeutic effect on arthritis by stimulating the biosynthesis of cartilage proteoglycan, and also increases the synthesis of cartilage-specific type II collagen, and has a hepatoprotective effect. Researches find that another GlcN derivative, acetylglucosamine GlcNAc, can induce K562 cells to differentiate towards macrophages, has obvious advantages in action concentration and action time compared with the traditional medicines, and is likely to become a novel cell differentiation induction medicine for clinical application because of small toxic and side effects.

The application of glucosamine in the food industry is as follows:

since GlcN is not toxic to human body, it is widely used as a food ingredient in both Japan and USA. Japan milk industry has begun selling GlcN-added milk beverage "Glucosamino Power" (family pack) since 2004 to 10 months, and Karl Pei, 2005 introduced

GlcN-added green tea beverage "FINESEPORT GLUCOSAMINE", other "GLUCOSAMINE soymilk" made from BAO JIU, and GlcN-added citrus beverage such as Dairy, Bojia, and ai Er Tai are also introduced into the market. Chenxin and the like research the antibacterial action of GlcN hydrochloride, and as a result, the found that the GlcN hydrochloride has obvious inhibition effect on common bacteria, mold and yeast in the tested food, wherein the bacteria are most sensitive, and the antibacterial action is gradually enhanced along with the increase of the concentration of the GlcN hydrochloride. Indicating that GlcN has broad prospects in becoming a novel natural food preservative.

Application of glucosamine in cosmetics

GlcNAc is a monomer of hyaluronic acid, is a basic composition unit of various important polysaccharides in organisms, and is also an additive of novel biochemical drugs, drug intermediates and high-grade cosmetics. It can enhance immunity and treat various inflammations. GlcNAc is an important precursor for the synthesis of bifidus factors and hyaluronic acid in the human body, and compared with hyaluronic acid, GlcNAc is a monomer with a small molecular weight, is very easily absorbed by the skin, and can provide nutrition, moisture and anti-free radicals when applied to cosmetics, so GlcNAc is often used for the production of high-grade cosmetics, and the addition amount of GlcNAc in the cosmetics is generally 0.1-5%. At present, various skin care products containing GlcNAc are available in domestic markets, the highest addition amount reaches 4%, and the effect is good.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention also aims to provide the saccharomyces cerevisiae and a method for producing the glucosamine by the saccharomyces cerevisiae, which optimize the fermentation process of the self-made engineering strains and improve the yield of the glucosamine.

To achieve these objects and other advantages in accordance with the present invention, there is provided a Saccharomyces cerevisiae H158-1C deposited at the China general microbiological culture Collection center with accession numbers: CGMCC No.11735, the preservation time is as follows: 11/25/2015.

A method for producing glucosamine by using saccharomyces cerevisiae comprises the following steps:

step one, seed culture: inoculating the saccharomyces cerevisiae on a slant culture medium containing kanamycin sulfate, culturing for 24-28 h at the temperature of 30-37 ℃, inoculating on a seed culture medium, and culturing to OD at the temperature of 30-37 ℃ and the rotating speed of 1100-2000 r/min₆₀₀When the value is 2-3, obtaining a seed culture solution;

step two, seed fermentation: inoculating the planting culture solution prepared in the second step onto a fermentation culture medium, and fermenting for 18-20 hours at the temperature of 35-40 ℃ and the rotating speed of 200-220 r/min to obtain a fermentation solution;

wherein the volume of the seed culture solution inoculated to the fermentation culture medium is 1-5% of the volume of the fermentation culture medium;

adding kanamycin sulfate with the final concentration of 70 mug/mL into the fermentation medium before inoculation, and controlling the residue to be 2.0%;

when OD of the bacterial body₆₀₀When the concentration is 0.6, 5g/L of lactose is added into the fermentation medium;

fermenting for 2-10 h in seeds and obtaining the OD of thalli₆₀₀When the cell growth rate is 2-3, the fermentation medium is fed with glucose in an initial exponential manner, and the cell growth rate is controlled to be 0.2h^-1；

The dissolved oxygen regulating strategy in seed fermentation is as follows: when the seeds are fermented for 0-2 h, 3-8 h, 9-12 h and 13-18 h, respectively controlling the dissolved oxygen amount to be 20%, 30%, 40% and 30%, the ventilation volume per ventilation to be 10vvm and the gas temperature to be 35-37 ℃;

and step three, purifying and crystallizing the fermentation liquor obtained in the step two to obtain the glucosamine.

Preferably, in the method for producing glucosamine by saccharomyces cerevisiae, in the first step, the slant culture medium comprises: 10-15 g/L of tryptone, 5-10 g/L, NaCl 10-15 g/L of yeast powder, 20-25 g/L of agar powder and 40-50 mg/L of kanamycin sulfate;

the pH value of the slant culture medium is 6.5-7.5, and the slant culture medium is sterilized at 0.1MPa for 25-30 min.

Preferably, in the method for producing glucosamine by saccharomyces cerevisiae, in the first step, the seed culture medium comprises: 50-60 g/L of D-glucosamine hydrochloride, 25-30 g/L of calcium carbonate, 10-15 g/L of monopotassium phosphate, 15-20 g/L of magnesium sulfate heptahydrate, 0-15 g/L of sodium nitrate, 10-15 g/L of yeast extract and 1-3 ml/L of corn steep liquor;

the pH value of the seed culture medium is 6.5-7.5, and the seed culture medium is sterilized at 0.1Mpa for 25-30 min.

Preferably, in the method for producing glucosamine by saccharomyces cerevisiae, in the second step, the fermentation medium comprises: 50-90 g/L of glucose, 10-20 g/L of lactose, 10-15 g/L of calcium carbonate, 15-20 g/L of monopotassium phosphate, 5-7 g/L of magnesium sulfate heptahydrate, 5-8 g/L of sodium nitrate, 5-10 g/L of yeast extract and 3-5 ml/L of corn steep liquor;

the pH value of the fermentation medium is 6.5-7.5, and the fermentation medium is sterilized at 0.1Mpa for 25-30 min.

Preferably, in the method for producing glucosamine by saccharomyces cerevisiae, kanamycin sulfate with a final concentration of 50-70 μ g/mL is added to the fermentation medium before inoculating the seed culture solution in the second step, and the residual amount of kanamycin sulfate is 20 μ g/mL.

Preferably, in the method for producing glucosamine by saccharomyces cerevisiae, the third step is specifically:

centrifuging the fermentation liquor prepared in the step two for 15-20 min at the rotating speed of 7500-8000 rpm, taking the supernatant, heating for 15-20 min at 90-100 ℃, centrifuging for 20-25 min at the rotating speed of 12000-13000 rpm, taking the supernatant to obtain stock solution,

decoloring the stock solution by using diatomite, adopting ammonium sulfate at the temperature of 30-35 ℃ as an eluent, stopping elution when the concentration of an ammonium sulfate solution is 0.7-1.0 mol/L to obtain an initial filtrate, adsorbing and filtering the initial filtrate by using styrene strong acid cationic resin, eluting by using absolute ethyl alcohol with the same volume as the initial filtrate when the sample introduction flow rate is 3.19BV/h and the elution flow rate is 3.8BV/h to obtain a final filtrate, standing the final filtrate at the temperature of 4 ℃ for 6-10 h, and performing suction filtration to obtain the glucosamine acid crystal.

The invention at least comprises the following beneficial effects: firstly, the invention is a microbial fermentation method for preparing glucosamine, is an important hexosamine, can effectively act on cartilage tissues to treat rheumatoid arthritis, is considered as a natural and harmless food and health-care product ingredient, and has wide market application prospect; secondly, the invention optimizes the fermentation process of the self-made engineering strain, determines that the yield of the glucosamine is obviously improved by implementing a lactose feeding strategy and a staged dissolved oxygen control strategy in the fermentation process, the intracellular and extracellular yields of the glucosamine are not less than 134.7g/L, the adsorption rate of the purification is not less than 95%, the elution rate is not less than 98%, the purity of the glucosamine is more than 98%, and the domestic advanced level is reached.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention

Drawings

FIG. 1 is a flow chart of glucosamine production by Saccharomyces cerevisiae according to the present invention.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Examples 1,

The Saccharomyces cerevisiae H158-1C is classified and named as Saccharomyces cerevisiae, and is preserved in China general microbiological culture Collection center with the preservation number of: CGMCC No.11735, the preservation time is as follows: 11 month and 25 days 2015; the address of the preservation unit is as follows: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

Examples 2,

As shown in FIG. 1, the present invention provides a method for producing glucosamine using Saccharomyces cerevisiae as described in example 1, comprising the steps of:

step one, seed culture: inoculating the Saccharomyces cerevisiae to a slant culture medium containing kanamycin sulfate, culturing at 30 deg.C for 24 hr, inoculating to a seed culture medium, and culturing at 30 deg.C and 1100r/min to OD₆₀₀When the value is 2, obtaining a seed culture solution;

step two, seed fermentation: inoculating the planting culture solution prepared in the second step onto a fermentation culture medium, fermenting for 18h at the temperature of 35 ℃ and the rotating speed of 200r/min,

wherein the volume of the seed culture solution inoculated on the fermentation culture medium is 1 percent of the volume of the fermentation culture medium;

fermenting for 2h in seed and the OD of thallus₆₀₀When the cell growth rate is 2, the fermentation medium is fed with glucose in an initial exponential manner, and the cell growth rate is controlled to be 0.2h^-1；

The dissolved oxygen regulating strategy in seed fermentation is as follows: when the seeds are fermented for 0-2 h, 3-8 h, 9-12 h and 13-18 h, respectively controlling the dissolved oxygen amount to be 20%, 30%, 40% and 30%, the ventilation volume of each ventilation to be 10vvm, and the gas temperature to be 35 ℃;

Wherein the content of the first and second substances,

in step one, the slant medium comprises: 10g/L of tryptone, 5g/L, NaCl 10g/L of yeast powder, 20g/L of agar powder and 40mg/L of kanamycin sulfate; the slant culture medium has pH of 6.5, and is sterilized at 0.1Mpa for 30 min.

In the first step, the seed culture medium comprises: 50g/L of D-glucosamine hydrochloride, 25g/L of calcium carbonate, 10g/L of monopotassium phosphate, 15g/L of magnesium sulfate heptahydrate, 0g/L of sodium nitrate, 10g/L of yeast extract and 1ml/L of corn steep liquor; the pH of the seed culture medium was 6.5, and sterilized at 0.1MPa for 25 min.

In the second step, the fermentation medium comprises: 50g/L of glucose, 10g/L of lactose, 10g/L of calcium carbonate, 15g/L of monopotassium phosphate, 5g/L of magnesium sulfate heptahydrate, 5g/L of sodium nitrate, 5g/L of yeast extract and 3ml/L of corn steep liquor; the pH of the fermentation medium was 6.5, and sterilized at 0.1MPa for 25 min.

In the second step, 50 mug/mL kanamycin sulfate is added to the fermentation medium before inoculating the seed culture solution, and the residual amount of the kanamycin sulfate is 20 mug/mL.

The third step is specifically as follows:

centrifuging the fermentation liquor prepared in the seed fermentation process at 7500rpm for 15min to remove cell thallus, collecting supernatant, heating at 90 deg.C for 15min, centrifuging at 12000rpm for 20min to remove impure protein in the supernatant, and collecting supernatant to obtain stock solution;

decolorizing the stock solution with diatomite, adopting ammonium sulfate at the temperature of 30 ℃ as an eluent, stopping elution when the concentration of an ammonium sulfate solution is 0.7mol/L to obtain an initial filtrate, adsorbing and filtering the initial filtrate by using styrene strong acid cation resin, eluting by using absolute ethyl alcohol with the same volume as the initial filtrate when the sample introduction flow rate is 3.19BV/h and the elution flow rate is 3.8BV/h to obtain a final filtrate, standing the final filtrate at the temperature of 4 ℃ for 6h, and performing suction filtration to obtain the glucosamine acid crystal, wherein the purity of the glucosamine acid crystal is more than 98%.

Examples 3,

The invention provides a method for producing glucosamine by using saccharomyces cerevisiae as described in embodiment 1, which is characterized by comprising the following steps:

step one, seed culture: inoculating the Saccharomyces cerevisiae onto slant culture medium containing kanamycin sulfate, culturing at 37 deg.C for 28 hr, inoculating onto seed culture medium, and culturing at 37 deg.C and 2000r/min to OD₆₀₀When the value is 3, obtaining a seed culture solution;

step two, seed fermentation: inoculating the planting culture solution prepared in the second step onto a fermentation culture medium, fermenting for 20h at the temperature of 40 ℃ and the rotating speed of 220r/min,

wherein the volume of the seed culture solution inoculated on the fermentation culture medium is 5 percent of the volume of the fermentation culture medium;

fermenting for 10h in seed and the OD of thallus₆₀₀When the angle is 3, the directionThe fermentation medium is added with glucose at the beginning of exponential flow, and the specific growth rate of the cells is controlled to be 0.2h^-1；

The dissolved oxygen regulating strategy in seed fermentation is as follows: when the seeds are fermented for 0-2 h, 3-8 h, 9-12 h and 13-18 h, respectively controlling the dissolved oxygen amount to be 20%, 30%, 40% and 30%, the ventilation volume of each ventilation to be 10vvm, and the gas temperature to be 37 ℃;

Wherein the content of the first and second substances,

in the first step, a slant culture medium contains 15g/L of tryptone, 10g/L, NaCl 15g/L of yeast powder, 25g/L of agar powder and 50mg/L of kanamycin sulfate; the slant culture medium has pH of 7.5, and is sterilized at 0.1MPa for 30 min.

In the first step, the seed culture medium comprises: 60g/L of D-glucosamine hydrochloride, 30g/L of calcium carbonate, 15g/L of monopotassium phosphate, 20g/L of magnesium sulfate heptahydrate, 15g/L of sodium nitrate, 15g/L of yeast extract and 3ml/L of corn steep liquor;

the pH of the seed culture medium was 7.5, and sterilized at 0.1MPa for 30 min.

In the second step, the fermentation medium comprises: 90g/L glucose, 20g/L lactose, 15g/L calcium carbonate, 20g/L potassium dihydrogen phosphate, 7g/L magnesium sulfate heptahydrate, 8g/L sodium nitrate, 10g/L yeast extract and 5ml/L corn steep liquor.

The third step is specifically as follows:

centrifuging the fermentation liquor prepared in the seed fermentation process at 8000rpm for 20min to remove cell thallus, collecting supernatant, heating at 100 deg.C for 15min, centrifuging at 13000rpm for 25min to remove impure protein in the supernatant, and collecting supernatant to obtain stock solution;

decolorizing the stock solution with diatomite, adopting ammonium sulfate at the temperature of 35 ℃ as an eluent, stopping elution when the concentration of an ammonium sulfate solution is 1.0mol/L to obtain an initial filtrate, adsorbing and filtering the initial filtrate by using a styrene strong acid cation resin, eluting by using absolute ethyl alcohol with the same volume as the initial filtrate when the sample introduction flow rate is 3.19BV/h and the elution flow rate is 3.8BV/h to obtain a final filtrate, standing the final filtrate at the temperature of 4 ℃ for 10h, and performing suction filtration to obtain an aminogluconic acid crystal; the purity of the glucosamine acid crystal is more than 98 percent.

Examples 4,

Screening of Saccharomyces cerevisiae

Fermentation strain: activating Saccharomyces Cerevisiae on a fresh plate culture medium, inoculating a loopful into a 10ml fermentation culture medium, culturing at 30 ℃ and at the rotating speed of 120rm, sampling every 24H, detecting the consumption of D-glucosamine and the generation condition of D-glucosamine by adopting paper chromatography to screen out a target strain capable of effectively oxidizing a substrate and accumulating the glucosamine, and after repeated screening for many times, the total yield of the glucosamine reaches 25g +/-1/L after fermenting for 24H +/-4H, and the strain is named as Saccharomyces Cerevisiae genetically engineered bacteria (Saccharomyces Cerevisiae) H158-1C (strain preservation number: CGMCC No: 11735).

II, preparing glucosamine

Seed culture: selecting a loop from a preserved and constructed saccharomyces cerevisiae 158-1C slant test tube by using an inoculating loop under the aseptic condition of an ultra-clean workbench, drawing a line on the surface of a slant culture medium containing kanamycin sulfate, placing the slant culture medium in a constant temperature incubator at 35-37 ℃ for inverted culture for 24-28 h, adopting a 250mL triangular flask for seed culture, filling 50mL seed culture medium into the triangular flask, selecting the saccharomyces cerevisiae into the triangular flask, culturing at 35 ℃, 1600r/min for 11h and OD (origin-destination) for 11h₆₀₀When the value is 3, a seed culture solution is obtained and taken out for standby.

Seed fermentation: inoculating the seed culture solution into a 500mL triangular flask (containing 50mL fermentation medium) according to the inoculation amount of 1-2% (v/v), and culturing at 37 deg.C for 210r/min until the thallus OD₆₀₀When the concentration reached 0.6, 5g/L lactose was added, kanamycin sulfate was added to the fermentation medium at a final concentration of 70. mu.g/mL before use, the residue was controlled to 2.0%, and the medium was cultured for 20 hours. Glucose feeding mode: no glucose is fed for 0-2 h; 2 to 10 hours and OD₆₀₀The glucose is added into the cell in an exponential manner, and the specific growth rate mu of the cell is controlled when the value is about 2.5_setIs 0.2h^-1. Dissolved oxygen regulation strategy:0-2 h, and the DO (dissolved oxygen) value is 20%; 3-8 h, and the DO value is 30%; 9-12 h, and the DO value is 40%; 13-18 h, the DO value is 30%, the ventilation volume is controlled to be 1.0vvm in the fermentation process, the temperature is 36 ℃, and the constant-speed feeding is carried out.

Purification and crystallization: centrifuging the fermentation liquid prepared in the seed fermentation process at 7700rpm for 18min to remove cell thallus, collecting supernatant, heating at 95 deg.C for 18min, centrifuging at 12500rpm for 23min to remove impure protein in the supernatant, collecting supernatant to obtain stock solution,

decolorizing the stock solution with diatomite, adopting ammonium sulfate at the temperature of 33 ℃ as an eluent, stopping elution when the concentration of an ammonium sulfate solution is 0.8mol/L to obtain an initial filtrate, adopting styrene strong acid cation resin for adsorption filtration of the initial filtrate, adopting absolute ethyl alcohol with the same volume as the initial filtrate for elution when the sampling flow rate is 3.19BV/h and the elution flow rate is 3.8BV/h to obtain a final filtrate, standing the final filtrate at the temperature of 4 ℃ for 8h, and performing suction filtration to obtain the glucosamine acid crystal. The adsorption rate was 95.43%, and the elution rate was 98.47%; the purity of the glucosamine acid crystal is more than 98 percent.

Wherein the slant culture medium comprises: 13g/L of tryptone, 7g/L, NaCl13g/L of yeast powder, 23g/L of agar powder and 45mg/L of kanamycin sulfate; the slant culture medium has pH of 7, and is sterilized at 0.1MPa for 30 min.

The seed culture medium comprises: 55g/L of D-glucosamine hydrochloride, 27g/L of calcium carbonate, 13g/L of monopotassium phosphate, 17g/L of magnesium sulfate heptahydrate, 13g/L of sodium nitrate, 13g/L of yeast extract and 2ml/L of corn steep liquor; the pH of the seed culture medium was 7, and sterilized at 0.1MPa for 28 min.

The fermentation medium comprises: 70g/L of glucose, 15g/L of lactose, 13g/L of calcium carbonate, 18g/L of monopotassium phosphate, 6g/L of magnesium sulfate heptahydrate, 7g/L of sodium nitrate, 8g/L of yeast extract and 4ml/L of corn steep liquor; the fermentation medium has pH of 7, and is sterilized at 0.1MPa for 28 min.

And (3) detection:

the detection method of intracellular GlcN comprises the following steps: adding 5.0mL of fermentation liquid into a 5mL centrifuge tube, centrifuging for 8min at 5000r/min, discarding the supernatant, washing the thalli with sterile water for 3 times, treating the thalli with 10mL of 6mol/L hydrochloric acid in a 100 ℃ water bath for 3h, neutralizing the thalli with 10mol/L NaOH to pH7.0, cooling the thalli on ice to room temperature, diluting to 50mL, adding 0.5mL of acetylacetone reagent into the thalli, treating the thalli in a 90 ℃ water bath for 1h, cooling the thalli to the room temperature, slowly adding 10mL of 96% (v/v) ethanol, then adding 1.0mL of DMAB (dimethylamino borane) reagent (DMAB reagent, weighing 1.6g of DMAB, dissolving the DMAB in 30mL 30m L concentrated hydrochloric acid and 30mL 96% ethanol), and uniformly mixing. After mixing, the mixture was left at room temperature for 1 hour, and colorimetric at 530nm, and intracellular GlcN yield was calculated according to the standard curve.

The method for detecting extracellular GlcN comprises the following steps: adding 5.0mL of fermentation liquor into a 5mL centrifuge tube, centrifuging at 8000r/min for 8min, adding 1.0mL of acetylacetone reagent into 0.5mL of centrifuged supernatant, treating in 90 ℃ water bath for 1h, cooling to room temperature, slowly adding 10mL of 96% (v/v) ethanol, then adding 1.0mL of DMAB reagent, and uniformly mixing. After mixing, the mixture was left at room temperature for 1 hour, and the extracellular GlcN yield was calculated from a standard curve by color comparison at 530 nm.

The yield of extracellular glucosamine is 7.21 +/-0.08 g/L, and the yield of intracellular glucosamine is 128.51 +/-0.09 g/L.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. The method for producing glucosamine by saccharomyces cerevisiae is characterized in that the saccharomyces cerevisiae is preserved in China general microbiological culture Collection center with the preservation number of: CGMCC No.11735, the preservation time is as follows: 11 month and 25 days 2015; the method comprises the following steps:

The dissolved oxygen regulating strategy in seed fermentation is as follows: when the seeds are fermented for 0-2 h, 3-8 h, 9-12 h and 13-18 h, respectively controlling the dissolved oxygen amount to be 20%, 30%, 40% and 30%, the ventilation volume of each ventilation to be 10vvm, and the gas temperature to be 35-37 ℃;

step three, purifying and crystallizing the fermentation liquor prepared in the step two to obtain glucosamine;

the third step is specifically as follows:

2. The method for producing glucosamine by saccharomyces cerevisiae according to claim 1, wherein in the first step, the slant culture medium comprises: 10-15 g/L of tryptone, 5-10 g/L, NaCl 10-15 g/L of yeast powder, 20-25 g/L of agar powder and 40-50 mg/L of kanamycin sulfate;

3. The method for producing glucosamine by saccharomyces cerevisiae according to claim 1, wherein in the first step, the seed culture medium comprises: 50-60 g/L of D-glucosamine hydrochloride, 25-30 g/L of calcium carbonate, 10-15 g/L of monopotassium phosphate, 15-20 g/L of magnesium sulfate heptahydrate, 0-15 g/L of sodium nitrate, 10-15 g/L of yeast extract and 1-3 ml/L of corn steep liquor;

4. The method for producing glucosamine by saccharomyces cerevisiae according to claim 1, wherein in the second step, the fermentation medium comprises: 50-90 g/L of glucose, 10-20 g/L of lactose, 10-15 g/L of calcium carbonate, 15-20 g/L of monopotassium phosphate, 5-7 g/L of magnesium sulfate heptahydrate, 5-8 g/L of sodium nitrate, 5-10 g/L of yeast extract and 3-5 ml/L of corn steep liquor;

5. The method for producing glucosamine by saccharomyces cerevisiae according to claim 1 or 4, wherein kanamycin sulfate is added to the fermentation medium in the second step to a final concentration of 50-70 μ g/mL before inoculating the seed culture solution, and the residual amount of kanamycin sulfate is 20 μ g/mL.