CN106608908B - Method for extracting glucose tolerance factor from chromium-containing yeast - Google Patents
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Abstract
The invention discloses a method for extracting a glucose tolerance factor from chromium-containing yeast. The method comprises the following steps: 1) extracting chromium-containing yeast powder with ammonia water to obtain an ammonia water extract; 2) and under the ice bath condition, extracting the ammonia water extracting solution by using an ethanol water solution, centrifuging, collecting supernate, and removing ethanol to realize the extraction of the glucose tolerance factor. The extraction method is based on the following principle: the compound of small molecular protein and chromium (GTF) can be dissolved in ammonia water and alcohol solution, while the inorganic chromium which does not produce biotransformation in thallus is not dissolved in alcohol solution or precipitated in ammonia water solution. Based on the characteristic of low GTF content in yeast, the invention adopts a method of combining ammonia water extraction and ethanol precipitation extraction, improves the GTF content in GTF extract, and reduces the content of impurity protein to a greater extent, thus being a method for effectively extracting GTF.
Description
Technical Field
The invention relates to a method for extracting a glucose tolerance factor, in particular to a method for extracting a glucose tolerance factor from chromium-containing yeast.
Background
Yeast-derived Glucose Tolerance Factor (GTF) is a complex of chromium and small molecular proteins (peptides), and trivalent organic chromium is an important active ingredient and an active center of the complex. GTF can strengthen insulin, promote glycometabolism and lipid metabolism, reverse glucose intolerance and improve glucose tolerance; protecting insulin B cells, and increasing the number and affinity of insulin receptors; improving fat metabolism, improving livestock and poultry production performance, improving ketone body quality and immune response and other physiological functions, and is widely applied to the fields of medicines, functional foods, feeds and the like.
Diabetes is a common chronic endocrine-metabolic disorder disease. Is a disease caused by partial or complete insulin loss, decreased living cell insulin receptors, or decreased receptor sensitivity. The trace element chromium, an indispensable trace metal, plays an important role in maintaining the homeostasis of blood glucose, and the absence of chromium or its biologically active form leads to glucose intolerance. The human body cannot synthesize chromium by itself and can only ingest chromium from food. In the United states and in some industrially developed countries, the dietary chromium intake does not reach the lower limit specified by ESADDI (50. mu.g/d).
At present, the types of chromium supplements used in diabetes research are many, and in addition to the classic GTF, chromium phenylalanine, chromium picolinate, chromium nicotinate, chromium tripropionate complex, chromium trichloride, and the like. GTF in yeast is the same as chromium hormone source in organism, is biological metalloprotein, and is safe. Saccharomyces cerevisiae is the most abundant source of GTF. During the fermentation process, chromium ions enriched in the yeast are not all part of GTF. Only metal ions in the environment are enriched through biological adsorption and biological conversion to generate GTF, and the GTF is converted into organic chromium; and the chromium which is adsorbed on the surface of the thallus and is not subjected to biotransformation is inorganic chromium. In the deep processing and scientific research of GTF, a method for effectively extracting GTF (organic chromium part) and simultaneously reducing hybrid protein to the maximum extent is important.
Disclosure of Invention
The invention aims to provide a method for extracting a glucose tolerance factor from chromium-containing yeast, and the extract obtained by the method has high content of total organic chromium and less protein impurities.
The method for extracting the glucose tolerance factor from the chromium-containing yeast comprises the following steps:
1) extracting chromium-containing yeast powder with ammonia water to obtain an ammonia water extract;
2) and under the ice bath condition, extracting the ammonia water extracting solution by using an ethanol water solution, centrifuging, collecting supernate, and removing ethanol to realize the extraction of the glucose tolerance factor.
In the method, in the step 1), the mesh number of the chromium-containing yeast powder is 20-200 meshes.
In the method, in the step 1), the extraction is performed at a temperature of 25-50 ℃ by adopting a shaking extraction manner, for example, at 37 ℃;
the oscillation frequency of the oscillation extraction is 100-400 rpm, such as 200 rpm;
the shaking extraction time is 60-300 minutes, such as 3 hours.
In the method, in the step 1), the concentration of the ammonia water is 0.05-0.25 mol/L, specifically 0.1 mol/L;
the dosage of the ammonia water is as follows: 10-50 mL of ammonia water is needed for 1g of the chromium-containing yeast powder, and 20mL of ammonia water is needed for 1g of the chromium-containing yeast powder.
In the above method, in step 2), the volume percentage concentration of the ethanol aqueous solution is 95%;
the adding amount of the ethanol aqueous solution is 15-70% of the volume of the ammonia water extracting solution, and specifically can be 15-60%, 15%, 30%, 40%, 50% or 60%.
In the method, in the step 2), the extraction time is 30-1200 minutes, specifically 30-60 minutes, 30 minutes or 60 minutes.
In the method, ethanol is removed in the step 2) by adopting a rotary vacuum concentration mode;
the conditions for the rotary vacuum concentration were as follows: the method is carried out at the rotating speed of 100-1000 r/min, the temperature is 30-60 ℃, the vacuum degree is 0.08MPa, for example, the method is carried out at the temperature of 50 ℃, the vacuum degree is 0.08MPa and the rotating speed of 800 r/min.
The method provided by the invention can be used for extracting the glucose tolerance factor in the yeast with high GTF content, wherein the yeast with high GTF content can be prepared by fermenting a strain YSI-3.7(CGMCC No 2687).
The extraction method is based on the following principle: the compound of small molecular protein and chromium (GTF) can be dissolved in ammonia water and alcohol solution, while the inorganic chromium which does not produce biotransformation in thallus is not dissolved in alcohol solution or precipitated in ammonia water solution. Based on the characteristic of low GTF content in yeast, the invention adopts a method of combining ammonia water extraction and ethanol precipitation extraction, improves the GTF content in GTF extract, and reduces the content of impurity protein to a greater extent, thus being a method for effectively extracting GTF.
Drawings
FIG. 1 is a schematic diagram showing the content changes of GTF and protein during microfiltration of the extract in example 2.
FIG. 2 is a purified GTF map of SuperdexG75 from example 2.
FIG. 3 is a gel chromatogram of Sephadex G25 in example 2.
FIG. 4 is a diagram showing RP-HPLC separation and purification in example 2.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The high GTF yeast used in the following examples was prepared by fermentation using YSI-3.7(CGMCC No2687) as follows:
liquid medium (YPD): 20g of glucose; 10g of yeast extract powder; 20g of soybean protein powder; 1L of distilled water; sterilizing at pH5.8, 121 deg.C for 20 min.
Seed liquid culture:
primary and secondary seed culture: the liquid content of the 250mL triangular flask was 50 mL. Inoculating 1mL of original strain YSI-3.7 frozen and preserved in glycerol into 50mLYPD culture medium, and culturing at 28 deg.C for 15 hr at rotation speed of 200 r/min; inoculating the strain into a 50mLYPD culture medium according to the inoculation amount of 10%, and culturing at the rotation speed of 200r/min and the culture temperature of 28 ℃ for 10-12 hours. A portion of the culture broth was taken before inoculation and examined under a microscope.
Fermenting the working fermentation liquor:
the liquid loading amount of a 50L fermentation tank for each fermentation is 30L, and the inoculation amount is 10%. YPD medium is used as fermentation working solution, wherein glucose and chromium solution are sterilized separately outside the tank (121 deg.C, 15min), yeast extract powder and soybean peptone are dissolved in the tank, and the tank is sterilized in situ (121 deg.C, 20 min). When in fermentation, glucose is aseptically added into the tank. Starting the stirrer and stirring uniformly. In the fermentation process, the rotating speed is connected with dissolved oxygen in series, the rotating speed is controlled by the dissolved oxygen, the dissolved oxygen is kept higher than 40 percent in the fermentation process, the culture temperature is 28 ℃, and the culture time is 44 hours.
The chromium solution adding mode is as follows:
adding chromium liquid once when fermentation is started, and feeding chromium for the second time: the initial chromium ion concentration of the fermentation broth was 400. mu.g/mL. When the fermentation time is 24 hours, the chromium solution is fed back, and the feeding is completed within 4 hours, so that the final chromium ion concentration in the fermentation liquid reaches 500 mug/mL.
After the fermentation is finished, the fermentation liquid is discharged from the tank, the fermentation liquid is collected, a tubular centrifuge is adopted for centrifugation (10000r/min), thalli are collected, deionized water is used for washing for three times, and freeze drying is carried out.
The prepared high GTF-containing yeast has the organic chromium content of 1234 mug/g thallus.
The protein content in the following examples was determined by the Bradford method.
In the following examples, the chromium extract was digested by microwave digestion, and the chromium content was measured by flame atomic absorption spectrometry (AA 6300 flame atomic absorption spectrophotometry) in accordance with GB/T15555.6-1995.
The microwave digestion method comprises the following steps:
pretreatment: placing GTF extractive solution in a sample cup, adding 6mL nitric acid, and placing on a heating plate at 160 deg.C for 30 min.
Microwave digestion: after the pretreatment solution was cooled slightly, 0.5mL of perchloric acid and 5mL (mass fraction) of ammonium persulfate solution were added thereto. The mixture was digested in a microwave digestion apparatus according to the digestion parameters shown in table 1.
TABLE 1 digestion parameter table of yeast microwave digestion instrument
Example 1 extraction of GTF from high GTF-containing Yeast
1) Pulverizing dried high chromium yeast (100 mesh), extracting with 0.1mol/L ammonia water solution at 37 deg.C and shaking rate of 200r/min for 3 hr. And then centrifuging the mixture for 30min at the rotation speed of 10000r/min, and collecting supernatant, namely the ammonia water extracting solution of GTF. Wherein the ratio of the yeast powder to the ammonia water solution is 1 g: 20 mL.
In the ammonia water extract of GTF obtained in the step, the organic chromium content is 5.80 mug/mL, the total organic chromium content is 145.07 mug, the protein content is 18.16mg/mL, and the total protein content is 181.62 mg. The chromium (μ g)/protein (mg) ratio was 0.80.
2) Measuring the volume of the ammonia water extract of GTF obtained in the step 1). And then placed in an ice bath. Under the ice bath condition, ethanol solution with the volume percentage concentration of 95% is respectively added into ammonia water extract according to the proportion of 60%, 50%, 40%, 30% and 15% and is kept stand for 30 minutes to precipitate the hybrid protein. Centrifuging for 30 minutes at 10000r/min, and respectively collecting supernatant and precipitate.
3) Taking the alcoholic extract with different concentrations obtained in the step 2) by volume. Then respectively carrying out rotary vacuum concentration to remove ethanol, wherein the vacuum concentration conditions are that the temperature is 50 ℃, the vacuum degree is 0.08MPa and the rotary speed is 800 r/min. Then carrying out vacuum freeze drying to obtain the extract containing GTF.
The protein content and the chromium content change in the supernatant in the different extraction methods in the steps 2) and 3) were dynamically monitored, and the extraction efficiency of chromium/protein in the extraction process was calculated, as shown in table 2.
TABLE 2 comparison of extraction efficiencies of different extraction methods
As can be seen from the data in Table 1, the ammonia extraction is matched with the 50% ethanol solution extraction, the total organic chromium content in the system is 101.13 mug, the protein content is 17.02 mug, the Cr/protein ratio is 5.94, and compared with the Cr/protein content of 0.8 and the protein content of 181.62 mug in the ammonia extracting solution, the chromium-protein ratio of the ammonia extraction matched with the 50% ethanol solution extraction method is improved by about 7.4 times; the content of the impure protein in the system is less, and the purification rate of the chromium protein is greatly improved.
Example 2 extraction from high GTF-containing Yeast and purification
1) Pulverizing dried high chromium yeast (the mesh number is 100), and extracting with 0.1mol/L ammonia water solution at 37 deg.C under shaking at a shaking frequency of 200r/min for 3 hr. Centrifuging at 10000r/min for 30min, and collecting supernatant as GTF ammonia water extractive solution. Wherein the ratio of the yeast powder to the ammonia water extraction solution is 1 g: 20 mL.
In the ammonia water extract of GTF obtained in the step, the organic chromium content is 5.80 mu g/ml, the total organic chromium content is 145.07 mu g, the protein content is 18.16mg/ml, the total protein content is 181.62mg, and the ratio of chromium (mu g)/protein (mg) is 0.80.
2) Measuring the volume of the ammonia water extract of GTF obtained in the step 1). And then placed in an ice bath. Under the ice bath condition, ethanol with the volume percentage concentration of 95 percent is added into the ammonia water extracting solution according to the proportion of 50 percent, the mixture is stood for 60 minutes for precipitating the hybrid protein, the mixture is centrifuged for 30 minutes under the condition of the rotating speed of 10000r/min, and the supernatant is collected.
3) Taking the alcoholic extract with different concentrations obtained in the step 2) by volume. Then concentrated in rotary vacuum to remove ethanol. The vacuum concentration condition is that the temperature is 50 ℃, the vacuum degree is 0.08MPa, and 800 r/min.
And (3) measuring the change of the protein content and the chromium content in the supernatant in the step 3), wherein the total organic chromium content in the extracting solution obtained in the step is 101.13 mu g, the total protein content is 17.02mg, and the chromium (mu g)/protein (mg) content is 5.94.
4) Carrying out 0.22 mu m membrane microfiltration on the ethanol extract of the GTF obtained in the step 3).
The change of GTF and protein in the extract before and after microfiltration is shown in figure 1, and as can be seen from figure 1, the content of GTF is not affected by the microfiltration process, and only impurity particles are removed.
5) Feeding gel chromatography Superdex G75 sample with specification of 1.6 × 80cm on the micro-filtrate obtained in step 4). The eluent is pH6.0, 50mM ammonia acetate buffer solution, and the elution rate is 0.5 mL/min. Collecting the eluent with the number of 20-30, namely the chromium peak in figure 2.
6) Concentrating the collected eluate, freezing at-20 deg.C, and vacuum freeze-drying to obtain GTF-containing extract.
7) Dissolving the extract obtained in step 6) with deionized water, and performing gel chromatography on gel chromatography Sephadex G25 column with specification of 1.6 × 80 cm. The eluent is deionized water, and the elution rate is 0.3 mL/min. Collecting the eluent with the number of 20-32, namely the chromium peak in figure 3.
8) Concentrating the collected eluate, freezing at-20 deg.C, and vacuum freeze-drying to obtain GTF-containing extract.
9) Dissolving the chromium peak eluate of the freeze-dried Sephadex G25 gel filtration chromatography with deionized water, and further purifying with a reversed-phase high performance liquid chromatography (RP-HPLC) preparative chromatographic column, wherein the elution conditions are shown in Table 2. Collecting the eluent, carrying out 254nm detection on the eluent, simultaneously carrying out microwave digestion, and determining the content of chromium in the eluent. The chromium peak was collected.
FIG. 4 is a HPLC purification profile.
TABLE 2 RP-HPLC elution conditions
And (3) concentrating the collected purified liquid in vacuum, freezing the concentrated liquid at the temperature of minus 20 ℃, and performing vacuum freeze drying to obtain a purified sample containing GTF, wherein the total organic chromium content is 25ug, the total protein content is 0.2mg, and the ratio of chromium (mu g)/protein (mg) is 125.
Claims (3)
1. A method for extracting glucose tolerance factor from chromium-containing yeast comprises the following steps:
1) extracting chromium-containing yeast powder with ammonia water to obtain an ammonia water extract;
the extraction is carried out at the temperature of 25-50 ℃ by adopting a vibration extraction mode;
the oscillation frequency of the oscillation extraction is 100-400 r/min;
the shaking extraction time is 60-300 minutes;
the concentration of the ammonia water is 0.05-0.25 mol/L;
the dosage of the ammonia water is as follows: 10-50 mL of ammonia water is needed for 1g of the chromium-containing yeast powder;
2) under the ice bath condition, extracting the ammonia water extracting solution by using an ethanol water solution, centrifuging, collecting supernate, and removing ethanol to realize the extraction of the glucose tolerance factor;
the volume percentage concentration of the ethanol aqueous solution is 95 percent;
the adding amount of the ethanol aqueous solution is 50% of the volume of the ammonia water extracting solution;
the extraction time is 30-1200 minutes;
the chromium-containing yeast is prepared by fermenting a strain YSI-3.7(CGMCC No 2687).
2. The method of claim 1, wherein: in the step 1), the mesh number of the chromium-containing yeast powder is 20-200 meshes.
3. The method according to claim 1 or 2, characterized in that: in the step 2), ethanol is removed by adopting a rotary vacuum concentration mode;
the conditions for the rotary vacuum concentration were as follows: the method is carried out at the rotating speed of 100-1000 r/min, the temperature is 30-60 ℃, and the vacuum degree is 0.08 MPa.
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| CN101045906A (en) * | 2006-03-30 | 2007-10-03 | 安琪酵母股份有限公司 | Chromium-rich saccharomyces cerevisiae, chromium-rich yeast product and their production process |
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| CN101045906A (en) * | 2006-03-30 | 2007-10-03 | 安琪酵母股份有限公司 | Chromium-rich saccharomyces cerevisiae, chromium-rich yeast product and their production process |
Non-Patent Citations (4)
| Title |
|---|
| Richard A Anderson等."Factors affecting the retention and extraction of yeast chromium".《Journal of Agric Food Chem》.1978,第26卷(第4期), * |
| ZetićVG等."Chromium uptake by Saccharomyces cerevisiae and isolation of glucose tolerance factor from yeast biomass".《J Biosci》.2001,第26卷(第2期), * |
| 刘鹭."高产葡萄糖耐量因子（Glucose Tolerance Factor）酵母菌的选育及其功能特性的研究".《中国博士学位论文全文数据库·基础科学辑》.2009,(第10期), * |
| 刘鹭等."酵母源性葡萄糖耐量因子(GTF)含量测定方法的研究".《食品与发酵工业》.2009,第35卷(第1期), * |
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