CN115975069A - High-purity water-soluble yeast beta-glucan and preparation method thereof - Google Patents
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Abstract
The invention belongs to the technical field of biological preparation, and particularly relates to high-purity water-soluble yeast beta-glucan and a preparation method thereof. The product has a purity of 99.5%, is easily soluble in water, has a weight average molecular weight of 9549-83621Da, has low and relatively uniform molecular weight, and has good bioactivity.
Description
Technical Field
The invention belongs to the technical field of biological preparation, and particularly relates to high-purity water-soluble yeast beta-glucan and a preparation method thereof.
Background
The yeast cell wall is a natural green food additive, contains various bioactive substances, namely beta-glucan, protein and mannan from inside to outside. Wherein the beta-glucan (beta-1, 3-glucan) has the functions of enhancing cell immunity, resisting cancer, resisting tumor, resisting radiation, controlling cholesterol, eliminating free radicals and the like. The beta-glucan can be divided into water (alkali) soluble beta-glucan and water (alkali) insoluble beta-glucan. Among beta-glucans derived from various sources, yeast cell wall-derived water-insoluble beta-glucan is proved to have excellent biological activity and is receiving wide attention, and beer yeast has been widely used in food industry and pharmaceutical industry as a safe model organism.
Yeast cells are composed of cell walls, cell membranes, vacuoles, nucleic acids and other small molecular substances, wherein the cell walls are the main sources for obtaining yeast beta-glucan. In the conventional cell wall extraction method, most of the products are mixtures containing unremoved amino acids, proteins, organelles and cells which are not completely decomposed, and the purity of the cell wall is influenced, so that the preparation process of the yeast beta-glucan is complicated. In the preparation process of the beta-glucan taking yeast cell walls as raw materials, common extraction methods such as an acid method, an enzyme method, an ultrasonic method, a high-pressure homogenization method and the like are difficult to effectively remove proteins and lipids in the yeast beta-glucan, and insoluble impurities such as residual gray matter cannot be removed, so that the purity of the yeast beta-glucan product is low. For example, the method for extracting the yeast beta-glucan provided by the patent with the publication number of CN113897294A takes waste beer yeast as a raw material, the process is complicated, the cost is high, and the purity of the obtained yeast beta-glucan is only 92.4% -93.5%.
In addition, the commonly obtained yeast beta-glucan has a special three-dimensional spiral structure, so that the yeast beta-glucan is difficult to dissolve in common solvents such as water, acid, alkali and the like, has a huge molecular weight, and greatly reduces the utilization rate of the yeast beta-glucan. The traditional methods for changing the yeast source water-insoluble beta-glucan into the yeast source water-insoluble beta-glucan are an acid method, an enzyme method, an oxidative degradation method, an ultrasonic degradation method and the like, and the purpose of solubility is achieved by degrading the long-chain structure of the polysaccharide into a short chain so as to reduce the molecular weight. Polysaccharides are high molecular weight compounds, the pure products of which are microscopically inhomogeneous, in general, pure polysaccharide actually refers to a homogeneous composition within a certain molecular weight range. The method has rigorous action conditions, the reaction process is not easy to control, the obtained product has larger molecular weight and is dispersed, the yeast beta-glucan contains oligosaccharide, disaccharide, polysaccharide consisting of more than ten molecular chains and the like, and the molecular weight distribution and polysaccharide uniformity of the yeast beta-glucan are seriously influenced; in addition, the technical means is relatively lagged, the process is immature, and the requirement of industrial production is difficult to meet. For example, patent publication No. CN101353383A, after hot water treatment and alkali treatment, the alkali extract is acid neutralized and high temperature leached to remove mannan, water soluble yeast beta-glucan is obtained, and simultaneously alkali extract precipitate is enzymolysis and water washing treated to obtain water insoluble yeast beta-glucan.
Therefore, a method for successfully converting water-insoluble yeast β -glucan into water-soluble yeast β -glucan and obtaining yeast β -glucan having high purity, small molecular weight and high uniformity is demanded to expand the application range of yeast β -glucan.
Disclosure of Invention
Aiming at the problems in the background technology, the invention provides the high-purity water-soluble yeast beta-glucan and the preparation method thereof, the prepared yeast beta-glucan product has the highest purity of 99.5 percent, is in a pure white thread shape, is easy to dissolve in water, and has the weight-average molecular weight of 9549-83621 Da.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a preparation method of high-purity water-soluble yeast beta-glucan comprises the following steps:
(1) Washing fresh waste yeast paste with water until the waste yeast paste does not contain beer flavor, and then sieving the waste yeast paste with a 80-mesh sieve to obtain cleaner yeast cells;
(2) Adding each g of wet yeast cells in the step (1) into 2.5mL of mixed solution containing 0.04M EDTA and 0.14M 2-mercaptoethanol, preserving the temperature for 15min at 30 ℃, and then centrifuging for 10min at 3000r/min to obtain a precipitate A; in the step, the sulfhydrylation treatment can lead the yeast cells to generate plasmolysis, thereby facilitating the later cracking of the yeast cells and the separation and extraction of the yeast cell walls.
(3) Adding each g of precipitate A into 4mL of 0.02M citric acid-phosphoric acid buffer (pH = 5.8) containing 30mg of helicase and 1M sorbitol, and incubating at 30 deg.C for 40-60min; snailase is targeted the yeast cells are lysed.
(4) Centrifuging the solution subjected to heat preservation in the step (3) at 6000 r/min for 10min to obtain a precipitate B, and adding water into the precipitate B according to the mass volume ratio of 1g;
(5) Sequentially adding density gradient media with the densities of 1.62g/mL, 1.55g/mL, 1.48g/mL and 1.41g/mL along the wall of the centrifugal tube, then slowly adding the suspension obtained in the step (4) into the uppermost layer, and centrifuging for 15-20min at a density gradient of 3000r/min in a horizontal centrifuge so as to separate and remove cell contents of yeast cells subjected to plasmolysis;
(6) Sucking the content of the second strip from top to bottom in the centrifugal tube after gradient centrifugation, washing with water for 2 times, centrifuging to obtain precipitate C, and drying the precipitate C to obtain pure yeast cell wall;
(7) Adding water with volume (unit mL) 60-80 times of the mass (unit g) of the yeast cell wall into the yeast cell wall to prepare suspension, and preserving heat for 4-8h in water bath at 50-90 ℃;
(8) Centrifuging the heat-insulated suspension to remove mannan and water-soluble protein in the suspension to obtain a precipitate D, centrifuging and washing the precipitate D, adding a NaOH solution with the concentration of 1-2%, wherein the mass-volume ratio of the precipitate D to the NaOH solution is 1g (10-30) mL, then carrying out water bath heat preservation at 60 ℃ for 2h, centrifuging to remove alkali-soluble yeast beta-glucan (or called water-soluble glucan which exists as an impurity in the technical scheme of converting the yeast source water-insoluble beta-glucan into soluble glucan), and simultaneously further removing residual mannan, water-soluble protein and lipid to obtain a precipitate E;
(9) Washing the precipitate E with water to neutrality, adding 1000U/g papain and 900U/g bromelain, keeping the temperature in 50 deg.C water bath for 6h, heating to inactivate enzyme, washing the treated product with water, and freeze drying;
(10) Adding dimethyl sulfoxide into the dried treated substance, heating for 2h to dissolve the treated substance in dimethyl sulfoxide, centrifuging at 8000 r/min, removing precipitate, and collecting supernatant; and (4) freeze-drying the supernatant to obtain the water (alkali) insoluble yeast beta-glucan.
(11) Adding ethanol into the supernatant of the step (10), wherein the volume of the ethanol is 5 times of that of the supernatant, standing, centrifuging at 6000 r/min, and removing the supernatant ethanol solution to obtain a precipitate F;
(12) Dissolving the precipitate F with distilled water, concentrating under reduced pressure, and collecting the concentrated solution;
(13) And (4) freeze-drying the concentrated solution to obtain the high-purity water-soluble yeast beta-glucan.
Preferably, the volume ratio of the density gradient medium to the suspension in the step (5) is 4; the density gradient medium is any one of sucrose, chlorinated brilliant or sodium chloride;
more preferably, the density gradient medium used in step (5) is sucrose.
Preferably, the mass-to-volume ratio of the treatment substance to the dimethyl sulfoxide in the step (10) is 1mg; the volume fraction of the dimethyl sulfoxide is 50-90%, and the dissolving temperature is 60-80 ℃.
More preferably, the volume fraction of dimethyl sulfoxide in the step (10) is 90%, and the dissolving temperature is 70 ℃.
Preferably, the concentration of ethanol in the step (11) is 40-100%; the standing time is 0.5-36h.
More preferably, the concentration of ethanol in the step (11) is 60%; the standing time is 36h.
The invention also discloses the high-purity water-soluble yeast beta-glucan prepared by the method.
Preferably, the high-purity water-soluble yeast beta-glucan has a purity of up to 99.5% and a weight-average molecular weight of 9549-83621 Da.
The invention has the beneficial effects that:
1. the method takes waste yeast paste as a raw material, firstly extracts water-insoluble yeast beta-glucan, and then converts the insolubility of the yeast beta-glucan into water solubility, and finally prepares the high-purity water-soluble yeast beta-glucan. The method adopts sulfhydrylation treatment to carry out plasmolysis on yeast cells (as shown in figure 2), and then adopts helicase to pertinently crack the yeast cells, and removes cell contents of the yeast cells subjected to the plasmolysis through density gradient centrifugation, thereby avoiding the situation that the protein, nucleic acid substances and cell wall fragments in the cells are tightly wound and difficult to remove due to the cell crushing by means of ultrasound, autolysis and the like. The separation process is simple and efficient, and has low requirements on equipment.
2. The method adopts low-temperature (50-90 ℃ and 100 ℃) water bath to remove the mannan in the yeast cell wall, avoids the safety problem and the energy problem caused by the conventional high-temperature extraction (100 ℃) in the industrialization, and saves the cost while removing a large amount of mannan. The method can easily remove the residual mannan, water-soluble protein and lipid in the water extraction process by using a small amount of alkali with the concentration of only 1-2% without using acid, and can detect that the effect of removing the water-soluble yeast beta-glucan is obvious by detecting the extracting solution by a phenol-sulfuric acid method under the condition of low alkali concentration (< 2%). On one hand, the production cost is reduced, the pollution is less, and the environment is friendly, and on the other hand, the damage of acid and alkali to the yeast beta-glucan structure is reduced.
3. The invention adopts dimethyl sulfoxide to assist in purification on the basis of removing protein by an enzyme method. The yeast cell wall is a dense mosaic structure formed by mannan-protein-beta-glucan. The literature indicates that the beta-glucan product obtained by purification by various methods has the purity of only 80-90 percent, and still contains impurities and proteins, probably because the enzyme method cannot degrade the beta-glucan product due to the tight connection structure of part of the proteins and the polysaccharides; the invention extracts the yeast beta-glucan by utilizing the property that the dimethyl sulfoxide can dissolve the water-insoluble yeast beta-glucan, and other insoluble substances become precipitates after centrifugation. Therefore, most of insoluble protein and gray matter doped in the raw material can be removed, and compared with the method which only adopts three steps of water extraction, alkali extraction and enzymolysis, the purity of the product is obviously improved. In addition, the dimethyl sulfoxide can be recycled after being recovered by a freeze dryer, so that the reagent utilization rate is effectively improved, and the process cost is reduced.
4. The method takes the waste yeast paste as a raw material, has low cost, is easy to obtain, has social benefit of waste utilization, utilizes dimethyl sulfoxide to assist a purified sample to effectively remove 94.60 percent of impurities in the sample obtained in the previous step, and combines ethanol extraction to finally obtain a high-purity water-soluble yeast beta-glucan product, wherein the highest conversion rate from water insolubility to water solubility of the yeast beta-glucan product is 42.86 percent, the highest purity of the yeast beta-glucan product is 99.5 percent, the weight average molecular weight of the yeast beta-glucan product is between 9549 and 83621Da, the molecular weight of the yeast beta-glucan product is low and relatively uniform, the utilization rate of the yeast beta-glucan product is improved, the improvement of the solubility of the yeast beta-glucan is promoted to a certain extent, and the obtained yeast beta-glucan product has good solubility and can be dissolved in solvents such as water, acid, alkali and the like at normal temperature; the yeast beta-glucan products extracted by the existing method mostly contain yeast beta-glucan with various molecular weights, but the invention can obtain the yeast beta-glucan products with single molecular weight. In addition, the yeast beta-glucan still has good biological activity after being converted into water-soluble, for example, the relative expression quantity of mRNA of IL-1 beta and IL-6 can be effectively reduced in cell inflammatory reaction, so that inflammatory factors are effectively eliminated, and a certain anti-inflammatory effect is achieved.
5. The preparation method has the advantages of simple and convenient steps, high operability, economic raw materials, low cost, environment friendliness, capability of recycling the used reagents, capability of successfully preparing the yeast beta-glucan product which has low molecular weight and high purity and is soluble in various reagents at normal temperature, simple process, high operability and low preparation cost, provides an effective reference for expanding the application range of the yeast beta-glucan, and is suitable for industrial production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a band diagram of the gradient after density gradient centrifugation in example 1.
FIG. 2 is an optical micrograph of yeast cells (a), plasmolyzed cells (b), and yeast cell walls (c) in example 2.
FIG. 3 is a graph showing a comparison between a dried sample (a) before dimethyl sulfoxide treatment, a dried sample (b) after dimethyl sulfoxide treatment, and an impurity (c) removed by dimethyl sulfoxide in example 2.
FIG. 4 is an appearance diagram of high-purity water-soluble yeast β -glucan in example 2.
FIG. 5 is a Fourier infrared spectrum of high purity water-soluble yeast beta-glucan in example 2.
FIG. 6 is a thin layer chromatogram of high purity water-soluble yeast β -glucan in example 2, wherein a is a glucose standard, b is a mannose standard, and c is the high purity water-soluble yeast β -glucan.
FIG. 7 is a graph showing the weight average molecular weight of high-purity water-soluble yeast β -glucan in example 2.
FIG. 8 shows the relative expression of mRNA of 4 inflammatory factors in macrophages of RAW264.7 mice stimulated by LPS in example 2.
FIG. 9 is a graph showing the effect of the weight average molecular weight of high purity water-soluble yeast β -glucan of examples 1 to 5 on solubility.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention are commercially available unless otherwise specified.
Example 1
A preparation method of high-purity water-soluble yeast beta-glucan comprises the following steps:
(1) Washing fresh waste yeast paste with water until the fresh waste yeast paste does not contain beer flavor, and sieving with a 80-mesh sieve to obtain cleaner yeast cells;
(2) Weighing 4g of yeast cells with wet weight in the step (1), adding the yeast cells into 10mL of mixed solution containing 0.04M EDTA and 0.14M 2-mercaptoethanol, preserving the heat at 30 ℃ for 15min, and then centrifuging at 3000r/min for 10min to obtain a precipitate A;
(3) Adding 4g of precipitate A into 16mL of 0.02M citric acid-phosphoric acid buffer (pH = 5.8) containing 120mg of helicase and 1M sorbitol, and incubating at 30 ℃ for 40min;
(4) Centrifuging the solution subjected to heat preservation in the step (3) at 6000 r/min for 10min to obtain a precipitate B, and adding water into the precipitate B according to the mass volume ratio of 1g;
(5) Sequentially adding cesium chloride solutions with the densities of 1.62, 1.55, 1.48 and 1.41g/mL along the wall of the centrifugal tube to serve as density gradient media, slowly adding the suspension in the step (4) to the uppermost layer, wherein the volume ratio of the density gradient media to the suspension is 4;
(6) As shown in fig. 1, sucking the contents of the second strip from top to bottom in the centrifuged centrifuge tube, washing with water for 2 times, centrifuging to obtain precipitate C, and drying the precipitate C to obtain pure dry yeast cell wall;
(7) Taking 20g of dry yeast cell walls, adding water with the volume being 80 times of the mass of the yeast cell walls into the dry yeast cell walls to prepare suspension, and carrying out water bath at 70 ℃ for heat preservation for 6 hours;
(8) Centrifuging the heat-preserved suspension at 6000 r/min for 10min, removing the supernatant to obtain a precipitate D, centrifuging and washing the precipitate D for 3 times, adding a NaOH solution with the concentration of 1.5%, wherein the mass-volume ratio of the precipitate D to the NaOH solution is 15mL, then oscillating in a water bath at 60 ℃ for heat preservation for 2h, centrifuging at 6000 r/min for 10min, and removing the dark red supernatant to obtain a precipitate E;
(9) Washing the precipitate E with water to neutrality, adding 1000U/g papain and 900U/g bromelain, keeping the temperature in 50 deg.C water bath for 6h, heating to inactivate enzyme, washing the treated product with water, and freeze drying;
(10) Taking 50 mg of dried treated substance, adding 10mL of dimethyl sulfoxide with volume fraction of 50%, heating and dissolving at 60 deg.C for 2h, centrifuging at 8000 r/min for 10min at high speed, discarding the precipitate of dark brown impurities, and collecting supernatant;
(11) Adding 40% ethanol into the supernatant, wherein the volume of the ethanol is 5 times of that of the supernatant, standing for 0.5h, centrifuging at 6000 r/min, and removing the supernatant ethanol solution to obtain precipitate F;
(12) Adding the precipitate F into distilled water for dissolving, then carrying out reduced pressure concentration, and collecting a concentrated solution;
(13) And (3) freeze-drying the concentrated solution at the temperature of-72 ℃ and under the condition of 7Pa to obtain the high-purity water-soluble yeast beta-glucan.
Detecting the cell wall extraction effect by a blood cell counting method, and measuring that the cell wall extraction rate is 98.2%; the solubility of the high purity water-soluble yeast beta-glucan (i.e., the conversion of the yeast beta-glucan from water-insoluble to water-soluble) was 36.2% as measured by phenol-sulfuric acid method; the purity of the high-purity water-soluble yeast beta-glucan is 95.84 percent by using a Congo red method; in addition, the weight average molecular weight of the high purity water-soluble yeast β -glucan was found to be 83621Da.
Example 2
A preparation method of high-purity water-soluble yeast beta-glucan comprises the following steps:
(1) Washing fresh waste yeast paste with water until the fresh waste yeast paste does not contain beer flavor, and sieving with a 80-mesh sieve to obtain cleaner yeast cells;
(2) Weighing 4g of wet yeast cells in the step (1), adding the yeast cells into 10mL of mixed solution containing 0.04M EDTA and 0.14M 2-mercaptoethanol, preserving the temperature for 15min at 30 ℃, and then centrifuging for 10min at 3000r/min to obtain a precipitate A; FIG. 2a is an optical photograph of normal yeast cells before the sulfhydrylation treatment, and FIG. 2b is an optical photograph of plasmolysis of the yeast cells after the sulfhydrylation treatment, showing that the plasmolysis occurred in the normal yeast cells after the successful sulfhydrylation treatment.
(3) Adding 4g of precipitate A into 16mL of 0.02M citric acid-phosphoric acid buffer (pH = 5.8) containing 120mg of helicase and 1M sorbitol, and incubating at 30 ℃ for 40min;
(4) Centrifuging the solution subjected to heat preservation in the step (3) at 6000 r/min for 10min to obtain a precipitate B, and adding water into the precipitate B according to the mass volume ratio of 1g;
(5) Sequentially adding sucrose solutions with the densities of 1.62, 1.55, 1.48 and 1.41g/mL along the wall of the centrifugal tube to serve as density gradient media, slowly adding the suspension in the step (4) to the uppermost layer, wherein the volume ratio of the density gradient media to the suspension is 4;
(6) And (3) sucking the contents of the second strip from top to bottom in the centrifuged centrifuge tube, washing with water for 2 times, centrifuging to obtain precipitate C, and drying the precipitate C to obtain pure dried yeast cell walls, wherein FIG. 2C shows the result of optical microscopy of the obtained yeast cell walls, and it can be seen that almost all yeast cells are cracked, and complete or semi-complete cell walls are remained.
(7) Weighing 20g of dry yeast cell walls, adding water with the volume 70 times of the mass of the yeast cell walls to prepare suspension, and carrying out water bath at 90 ℃ for heat preservation for 8h;
(8) Centrifuging the heat-preserved suspension at 6000 r/min for 10min, removing the supernatant to obtain a precipitate D, centrifuging and washing the precipitate D for 3 times, then adding a NaOH solution with the concentration of 1.5%, wherein the mass-volume ratio of the precipitate D to the NaOH solution is 15mL, then oscillating in a water bath at 60 ℃ for heat preservation for 2h, centrifuging at 6000 r/min for 10min, and discarding the dark red supernatant to obtain a precipitate E.
At this time, 1.5% NaOH solution was added to the precipitate E at a mass-to-volume ratio of 1g to 15mL, followed by shaking in a water bath at 60 ℃ for 2 hours and centrifugation at 6000 r/min for 10 minutes, and the supernatant was collected and examined by the [ phenol sulfuric acid method ] to find that there was no UV absorption, indicating that there was no sugar in the supernatant, indicating that the water-soluble yeast β -glucan had been completely removed and that the precipitate E contained no water-soluble yeast β -glucan.
(9) Washing the precipitate E with water to neutrality, adding 1000U/g papain and 900U/g bromelain, keeping the temperature in 50 deg.C water bath for 6h, heating to inactivate enzyme, washing the treated product with water, and freeze drying;
(10) Taking 50 mg of dried treated substance, adding 10mL of dimethyl sulfoxide with volume fraction of 50%, heating and dissolving at 70 deg.C for 2h, centrifuging at 8000 r/min for 10min at high speed, discarding the precipitate of tawny impurity, and collecting supernatant; the supernatant was freeze-dried to obtain insoluble yeast β -glucan as shown in FIG. 3 b.
(11) Adding 60% ethanol into the supernatant, wherein the volume of the ethanol is 5 times of that of the supernatant, standing for 36h, centrifuging at 6000 r/min, and removing the supernatant ethanol solution to obtain precipitate F;
(12) Adding the precipitate F into distilled water for dissolving, then carrying out reduced pressure concentration, and collecting a concentrated solution;
(13) The concentrated solution was freeze-dried at-72 ℃ under 7Pa to obtain a high-purity water-soluble yeast β -glucan as shown in FIG. 3, which was white-filamentous in appearance as shown in FIG. 4.
(1) Yeast cell wall yield assay
And (3) determining the yeast cell wall obtained after the yeast cell is cracked by adopting a cell counting method:
the yield of yeast cell walls was =1- (number of cells after treatment/number of cells before treatment) =99.5%.
(2) And (3) measuring the purification effect of dimethyl sulfoxide:
FIG. 3a shows the dried sample before the DMSO treatment (i.e., the treated product after drying in step (3)), FIG. 3b shows the dried sample after the DMSO treatment (i.e., the insoluble yeast β -glucan obtained by freeze-drying the supernatant in step (4)), and FIG. 3c shows the impurities removed by the DMSO treatment, and comparing FIGS. 3a-c, it can be seen that the DMSO significantly removed most of the colored impurities in the sample, making the color of the sample more white and clean.
Further determining the impurity removal effect of dimethyl sulfoxide in the method, specifically: weighing the mass of impurities removed in the dimethyl sulfoxide treatment, detecting the content of impurities in the dried sample before dimethyl sulfoxide treatment (i.e. the treated product dried in step (3)) by Congo red method, repeating the measurement for three times, and averaging, corresponding to M 1 And M 2 Substituted into the following formulaAnd (3) obtaining the impurity removal rate of dimethyl sulfoxide treatment, namely:
removal rate = (M) 1 / M 2 )×100%=(0.0298g/0.0315g)×100%=94.60%
From the above formula results, it can be seen that the sample was purified with dmso-assisted to remove 94.60% of impurities.
The impurity components were detected by kjeldahl method, and the results showed that 87.3% of the impurities were proteins, and the remaining 12.7% could be lipids or grey matter.
(3) Substance identification and analysis of physicochemical properties:
the substance identification of the high purity water-soluble yeast β -glucan obtained in this example was carried out by fourier infrared spectroscopy, and the results are shown in fig. 5. In FIG. 5, at 3392.96cm -1 Has a wider and larger absorption peak which is the stretching vibration absorption peak of O-H and is 2920.68cm -1 The absorption peak is the stretching vibration peak of methylene C-H; and 1638.99cm -1 And 1078.70cm -1 -1042.00cm -1 The points are respectively absorption peaks of bending vibration and stretching vibration of C = O; at 1259.76cm -1 Is the common resultant absorption peak of bending and stretching vibrations of-OH; at 1419.35cm -1 Is sub-CH 2 Bending vibration of (2); at 891.72cm -1 The absorption peak at (a) indicates that the glycosidic bond configuration is the beta configuration. Therefore, the compound can be basically judged to be the beta-configuration glycoside compound.
The monosaccharide composition of the high-purity water-soluble yeast β -glucan product was measured by thin layer chromatography, and the results are shown in fig. 6, in which a is a glucose standard, b is a mannose standard, and c is the product, and it was found that the monosaccharide composition of the product was glucose, and it was further determined that the product was glucan polymerized from glucose monosaccharide.
The physical properties of the high-purity water-soluble yeast β -glucan were analyzed, and the results are shown in table 1.
TABLE 1 analysis of physical Properties
Note: "+" represents soluble, "-" represents insoluble.
As is clear from Table 1, the high-purity water-soluble yeast β -glucan is soluble in common solvents such as water, acids and alkalis at ordinary temperature.
(4) And (3) determination of purity and dissolution rate:
the purity of the yeast beta-glucan is measured by a Congo red method, and specifically comprises the following steps: weighing a certain amount of the yeast beta-glucan prepared in the embodiment as a sample, dissolving the sample with 0.1 mol/L phosphate buffer solution with pH 7.5, reacting the dissolved sample with 0.01% Congo red solution, and then measuring absorbance; the absorbance was measured with no sample as a control to obtain a standard equation. Substituting the obtained light absorption value into a standard equation to obtain the content of the yeast beta-glucan in the sample, and substituting into the following formula to obtain the purity of the yeast beta-glucan:
purity of yeast β -glucan = (content value/weighed value) × 100% =99.5%.
Performing element analysis by using a semi-macroelement analyzer to obtain that the content of the N element in the yeast beta-glucan product is 0.41 percent; the water soluble solubility (i.e., conversion) of the yeast β -glucan was 42.86% as measured by the phenol-sulfuric acid method using the following formula:
solubility = (measured value/weighed value) × 100%.
(5) Weight average molecular weight
The molecular weight distribution of the low yeast β -glucan was measured by using a high performance liquid chromatograph (exclusion column, differential detector), and the results are shown in fig. 7. As can be seen from FIG. 7, yeast β -glucan molecules having weight average molecular weights of 31622Da and 9549Da, respectively, are present in the yeast β -glucan.
(6) Anti-inflammatory activity:
RAW264.7 cells (mouse monocyte macrophage leukemia cells) in the logarithmic growth phase were digested, centrifuged, resuspended in DMEM complete medium (containing 10% serum and 1% double antibody), and resuspended at 1X 10 5 Density of/mL was seeded in 6-well plates and CO was% 2 The culture was continued overnight for 24 h. The final concentration of the solution was 1. Mu.g/mL -1 The inflammation model was established with LPS (Amersham pharmacia Biotech), and the test group was set as a positive control group, to which LPS (1. Mu.g.mL) was added -1 ) And the high purity water prepared in this exampleSoluble yeast beta-glucan (20, 40, 60, 80, 100. Mu.g. ML) -1 ) The blank group was supplemented with normal DMEM complete medium. The relative expression levels (defined as fold change) of the genes of interest (IL-6, IL-1. Beta.) were determined using 2- △△CT And (4) determining the method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene, and expression levels were normalized to the fold change detected in the corresponding control cells (defined as 1.0). The results are shown in FIG. 8: compared with LPS action group, after adding beta-glucan, the inflammation related factors IL-6 and IL-1 beta expression is reduced, which shows that the beta-glucan has certain inhibition effect on LPS induced inflammation.
(7) And (3) recovering a reagent:
and (4) freeze-drying the centrifuged supernatant in the step (10) to temporarily retain dimethyl sulfoxide in a freeze dryer through physical state change, discharging the dimethyl sulfoxide from a liquid discharge port after freeze-drying is finished, collecting the discharged dimethyl sulfoxide, and continuously recycling.
Example 3
A preparation method of high-purity water-soluble yeast beta-glucan comprises the following steps:
(1) Washing fresh waste yeast paste with water until the fresh waste yeast paste does not contain beer flavor, and sieving with a 80-mesh sieve to obtain cleaner yeast cells;
(2) Weighing 4g of wet yeast cells in the step (1), adding the yeast cells into 10mL of mixed solution containing 0.04M EDTA and 0.14M 2-mercaptoethanol, preserving the temperature for 15min at 30 ℃, and then centrifuging for 10min at 3000r/min to obtain a precipitate A.
(3) Adding 4g of precipitate A into 16mL of 0.02M citric acid-phosphoric acid buffer (pH = 5.8) containing 120mg of helicase and 1M sorbitol, and incubating at 30 ℃ for 40min;
(4) Centrifuging the solution after heat preservation in the step (3) at 6000 r/min for 10min to obtain a precipitate B, and adding water into the precipitate B according to a mass volume ratio of 1g;
(5) Sequentially adding sodium chloride solutions with the densities of 1.62, 1.55, 1.48 and 1.41g/mL along the wall of the centrifugal tube to serve as density gradient media, slowly adding the suspension in the step (4) to the uppermost layer, wherein the volume ratio of the density gradient media to the suspension is 4;
(6) Sucking the contents of the second strip from top to bottom in the centrifuged centrifuge tube, washing with water for 2 times, centrifuging to obtain precipitate C, and drying the precipitate C to obtain pure dry yeast cell wall;
(7) Weighing 20g of dry yeast cell walls, adding water with the volume 60 times of the mass of the yeast cell walls to prepare suspension, and carrying out water bath heat preservation at 50 ℃ for 4 hours;
(8) Centrifuging the heat-preserved suspension at 6000 r/min for 10min, removing the supernatant to obtain a precipitate D, centrifuging and washing the precipitate D for 3 times, adding a NaOH solution with the concentration of 1.5%, wherein the mass-volume ratio of the precipitate D to the NaOH solution is 15mL, then oscillating in a water bath at 60 ℃ for heat preservation for 2h, centrifuging at 6000 r/min for 10min, and removing the dark red supernatant to obtain a precipitate E;
(9) Washing the precipitate E with water to neutrality, adding 1000U/g papain and 900U/g bromelin, keeping the temperature in 50 deg.C water bath for 6h, heating to inactivate enzyme, washing the treated product with water, and freeze drying;
(10) Taking 50 mg of dried treated substance, adding 10mL of dimethyl sulfoxide with volume fraction of 50%, heating and dissolving at 80 deg.C for 2h, centrifuging at 8000 r/min for 10min at high speed, discarding the precipitate of dark brown impurities, and collecting supernatant;
(11) Adding 100% ethanol into the supernatant, wherein the volume of ethanol is 5 times of the volume of the supernatant, standing for 12 h, centrifuging at 6000 r/min, and discarding the supernatant ethanol solution to obtain precipitate F;
(12) Adding the precipitate F into distilled water for dissolving, then carrying out reduced pressure concentration, and collecting a concentrated solution;
(13) And (3) freeze-drying the concentrated solution at-72 ℃ and 7Pa to obtain the high-purity water-soluble yeast beta-glucan.
Detecting the cell wall extraction effect by a blood cell counting method, wherein the extraction rate of the yeast cell wall in the embodiment is 97.8 percent; the solubility (namely the conversion rate) of the high-purity water-soluble yeast beta-glucan is 39.84 percent by adopting a phenol-sulfuric acid method; the purity of the water-soluble yeast beta-glucan is measured to be 92.58 percent by utilizing a Congo red reagent prepared in advance; in addition, the weight average molecular weight of the high purity water-soluble yeast β -glucan was measured to be 60963Da and 20264Da.
Example 4
A preparation method of high-purity water-soluble yeast beta-glucan comprises the following steps:
(1) Washing fresh waste yeast paste with water until the fresh waste yeast paste does not contain beer flavor, and sieving with a 80-mesh sieve to obtain cleaner yeast cells;
(2) Weighing 4g of wet yeast cells in the step (1), adding the yeast cells into 10mL of mixed solution containing 0.04M EDTA and 0.14M 2-mercaptoethanol, preserving the temperature for 15min at 30 ℃, and then centrifuging for 10min at 3000r/min to obtain a precipitate A.
(3) Adding 4g of precipitate A into 16mL of 0.02M citric acid-phosphoric acid buffer (pH = 5.8) containing 120mg of helicase and 1M sorbitol, and incubating at 30 deg.C for 50min;
(4) Centrifuging the solution subjected to heat preservation in the step (3) at 6000 r/min for 10min to obtain a precipitate B, and adding water into the precipitate B according to the mass volume ratio of 1g;
(5) Sequentially adding cesium chloride solutions with the densities of 1.62, 1.55, 1.48 and 1.41g/mL along the wall of the centrifugal tube to serve as density gradient media, slowly adding the suspension in the step (4) to the uppermost layer, wherein the volume ratio of the density gradient media to the suspension is 4;
(6) Sucking the contents of the second strip from top to bottom in the centrifuged centrifuge tube, washing with water for 2 times, centrifuging to obtain precipitate C, and drying the precipitate C to obtain pure dry yeast cell wall;
(7) Taking 20g of dry yeast cell walls, adding water with the volume 80 times of the mass of the yeast cell walls to prepare suspension, and preserving heat for 6 hours in 70 ℃ water bath;
(8) Centrifuging the heat-preserved suspension at 6000 r/min for 10min, removing the supernatant to obtain a precipitate D, centrifuging and washing the precipitate D for 3 times, then adding 1% NaOH solution, wherein the mass-volume ratio of the precipitate D to the NaOH solution is 110 mL, oscillating in a water bath at 60 ℃ for heat preservation for 2h, centrifuging at 6000 r/min for 10min, and removing a dark red supernatant to obtain a precipitate E;
(9) Washing the precipitate E with water to neutrality, adding 1000U/g papain and 900U/g bromelin, keeping the temperature in 50 deg.C water bath for 6h, heating to inactivate enzyme, washing the treated product with water, and freeze drying;
(10) Taking 50 mg of dried treated substance, adding 10mL of dimethyl sulfoxide with volume fraction of 50%, heating and dissolving at 60 deg.C for 2h, centrifuging at 8000 r/min for 10min at high speed, discarding the precipitate of dark brown impurities, and collecting supernatant;
(11) Adding 40% ethanol into the supernatant, wherein the volume of the ethanol is 5 times of that of the supernatant, standing for 36h, centrifuging at 6000 r/min, and removing the supernatant ethanol solution to obtain precipitate F;
(12) Adding the precipitate F into distilled water for dissolving, then carrying out reduced pressure concentration, and collecting a concentrated solution;
(13) And (3) freeze-drying the concentrated solution at-72 ℃ and 7Pa to obtain the high-purity water-soluble yeast beta-glucan.
Detecting the cell wall extraction effect by a blood cell counting method, and determining that the cell wall extraction rate is 96.5%; the solubility (namely the conversion rate) of the high-purity water-soluble yeast beta-glucan is 41.73 percent by adopting a phenol-sulfuric acid method; the purity of the high-purity water-soluble yeast beta-glucan is 94.36 percent by a Congo red method; in addition, the weight average molecular weight of the high purity water-soluble yeast β -glucan was measured to be 40369Da.
Example 5
A preparation method of high-purity water-soluble yeast beta-glucan comprises the following steps:
(1) Washing fresh waste yeast paste with water until the fresh waste yeast paste does not contain beer flavor, and sieving with a 80-mesh sieve to obtain cleaner yeast cells;
(2) Weighing 4g of wet yeast cells in the step (1), adding the yeast cells into 10mL of mixed solution containing 0.04M EDTA and 0.14M 2-mercaptoethanol, preserving the temperature for 15min at 30 ℃, and then centrifuging for 10min at 3000r/min to obtain a precipitate A.
(3) Adding 4g of precipitate A into 16mL of 0.02M citric acid-phosphoric acid buffer (pH = 5.8) containing 120mg of helicase and 1M sorbitol, and incubating at 30 deg.C for 60min;
(4) Centrifuging the solution subjected to heat preservation in the step (3) at 6000 r/min for 10min to obtain a precipitate B, and adding water into the precipitate B according to the mass volume ratio of 1g;
(5) Sequentially adding cesium chloride solutions with the densities of 1.62, 1.55, 1.48 and 1.41g/mL along the wall of the centrifugal tube to serve as density gradient media, slowly adding the suspension in the step (4) to the uppermost layer, wherein the volume ratio of the density gradient media to the suspension is 4;
(6) Sucking the contents of the second strip from top to bottom in the centrifuged centrifuge tube, washing with water for 2 times, centrifuging to obtain precipitate C, and drying the precipitate C to obtain pure dry yeast cell wall;
(7) Taking 20g of dry yeast cell walls, adding water with the volume 80 times of the mass of the yeast cell walls to prepare suspension, and preserving heat for 6 hours in 70 ℃ water bath;
(8) Centrifuging the heat-preserved suspension at 6000 r/min for 10min, removing the supernatant to obtain a precipitate D, centrifuging and washing the precipitate D for 3 times, adding a NaOH solution with the concentration of 2%, wherein the mass-volume ratio of the precipitate D to the NaOH solution is 1g;
(9) Washing the precipitate E with water to neutrality, adding 1000U/g papain and 900U/g bromelain, keeping the temperature in 50 deg.C water bath for 6h, heating to inactivate enzyme, washing the treated product with water, and freeze drying;
(10) Taking 50 mg of dried treated substance, adding 10mL of dimethyl sulfoxide with volume fraction of 50%, heating and dissolving at 60 deg.C for 2h, centrifuging at 8000 r/min for 10min at high speed, discarding the precipitate of dark brown impurities, and collecting supernatant;
(11) Adding 80% ethanol into the supernatant, wherein the volume of ethanol is 5 times of the volume of the supernatant, standing for 24 hr, centrifuging at 6000 r/min, and discarding the supernatant ethanol solution to obtain precipitate F;
(12) Adding the precipitate F into distilled water for dissolving, then carrying out reduced pressure concentration, and collecting a concentrated solution;
(13) And (3) freeze-drying the concentrated solution at the temperature of-72 ℃ and under the condition of 7Pa to obtain the high-purity water-soluble yeast beta-glucan.
Detecting the cell wall extraction effect by a blood cell counting method, and measuring that the cell wall extraction rate is 97.4%; the solubility (namely the conversion rate) of the high-purity water-soluble yeast beta-glucan is 40.52 percent by adopting a phenol-sulfuric acid method; the purity of the high-purity water-soluble yeast beta-glucan is 95.23 percent by using a Congo red method; in addition, the weight average molecular weight of the high purity water-soluble yeast β -glucan was found to be 53108Da.
The correspondence between the solubility and the weight average molecular weight of the high purity water-soluble yeast β -glucan produced in examples 1 to 5 is shown in table 2, and the influence of the weight average molecular weight on the solubility is shown in fig. 9.
TABLE 2 corresponding relationship between solubility and weight average molecular weight
As can be seen from Table 2 and FIG. 9, the solubility of the yeast β -glucan product of the present invention tends to decrease as the weight average molecular weight thereof gradually increases. It is shown that the solubility of yeast beta-glucan may have a certain relationship with the weight average molecular weight. This is also consistent with the reports in the literature [ yeast β -glucan solubility and emulsifiability improvement and application studies ]. The weight average molecular weight of the yeast beta-glucan prepared by the invention is generally smaller and the uniformity is higher, so that the utilization rate of the yeast beta-glucan is improved, and the improvement of the solubility of the yeast beta-glucan is promoted to a certain extent.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A preparation method of high-purity water-soluble yeast beta-glucan is characterized by comprising the following steps:
(1) Mixing EDTA and 2-mercaptoethanol, adding waste yeast paste, maintaining the temperature for the first time, and centrifuging to obtain precipitate A;
(2) Dissolving helicase and sorbitol in citric acid-phosphoric acid buffer solution to obtain mixed solution, adding precipitate A, performing second heat preservation, centrifuging to obtain precipitate B, and adding water into precipitate B to obtain suspension;
(3) Adding a density gradient medium into a centrifugal tube, then adding the suspension obtained in the step (2) for density gradient centrifugation, collecting the content of a second strip from top to bottom, washing with water, centrifuging to obtain a precipitate C, and drying the precipitate C to obtain a yeast cell wall;
(4) Adding water into the yeast cell wall obtained in the step (3), carrying out heat preservation for the third time, centrifuging to obtain a precipitate D, then adding the precipitate D into a NaOH solution, carrying out heat preservation for the fourth time, centrifuging to obtain a precipitate E, and carrying out enzyme treatment on the precipitate E to obtain a treated substance;
(5) Dissolving the treated substance in the step (4) in dimethyl sulfoxide, centrifuging, and collecting supernatant;
(6) And (5) adding ethanol into the supernatant obtained in the step (5), standing, centrifuging to obtain a precipitate E, adding water to dissolve the precipitate E, concentrating, and drying to obtain the high-purity water-soluble yeast beta-glucan.
2. The method for producing high-purity water-soluble yeast β -glucan according to claim 1, wherein: the mass-to-volume ratio of the precipitate A to the mixed solution in the step (2) is 1g; the concentration of the helicase in the mixed solution was 7.5 mg/mL, the concentration of sorbitol was 1M, the concentration of the citric acid-phosphate buffer was 0.02M, pH =5.8; the second heat preservation temperature is 30 ℃, and the time is 40-60min; the mass-to-volume ratio of precipitate B to water was 1g.
3. The method for producing a high-purity water-soluble yeast β -glucan according to claim 1, characterized in that: the density of the density gradient medium in the step (3) is 1.62g/mL, 1.55g/mL, 1.48g/mL and 1.41g/mL, and the density gradient medium is any one of sucrose, cesium chloride or sodium chloride; the volume ratio of the density gradient medium to the suspension is 4; the rotating speed of the density gradient centrifugation is 3000r/min, and the time is 15-20min.
4. The method for producing a high-purity water-soluble yeast β -glucan according to claim 1, characterized in that: the mass volume ratio of the yeast cell wall to the water in the step (4) is 1g (60-80) mL; the temperature for the third heat preservation is 50-90 ℃ and the time is 4-8h.
5. The method for producing a high-purity water-soluble yeast β -glucan according to claim 4, wherein: the mass volume ratio of the precipitate D to the NaOH solution is 1g (10-30) mL; the concentration of the NaOH solution is 1% -2%; the temperature of the fourth heat preservation is 60 ℃ and the time is 2h.
6. The method for producing high-purity water-soluble yeast β -glucan according to claim 1, wherein: the mass-to-volume ratio of the treatment substance in the step (5) to the dimethyl sulfoxide is 1mg; the volume fraction of the dimethyl sulfoxide is 50-90 percent; the dissolving temperature is 60-80 ℃.
7. The method for producing high-purity water-soluble yeast β -glucan according to claim 1, wherein: the volume ratio of the supernatant to the ethanol in the step (6) is 1; the concentration of the ethanol is 40% -100%; the standing time is 0.5-36h.
8. A high purity water soluble yeast β -glucan produced by the method of any one of claims 1 to 7.
9. The high purity water soluble yeast beta-glucan according to claim 6, wherein: the purity of the high-purity water-soluble yeast beta-glucan is 99.5 percent at most, and the weight-average molecular weight is 9549-83621 Da.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101353383A (en) * | 2008-09-17 | 2009-01-28 | 山东京博控股发展有限公司 | Water-soluble yeast beta-dextran and preparation thereof |
| CN101560269A (en) * | 2009-05-22 | 2009-10-21 | 广东药学院 | Method for preparing yeast beta-1, 3-glucan |
| CN105255964A (en) * | 2015-10-30 | 2016-01-20 | 宁波希诺亚海洋生物科技有限公司 | Production method of beta-glucan |
| CN115491311A (en) * | 2022-09-05 | 2022-12-20 | 武汉华美生物工程有限公司 | Method for crushing cell wall of pichia pastoris and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101353383A (en) * | 2008-09-17 | 2009-01-28 | 山东京博控股发展有限公司 | Water-soluble yeast beta-dextran and preparation thereof |
| CN101560269A (en) * | 2009-05-22 | 2009-10-21 | 广东药学院 | Method for preparing yeast beta-1, 3-glucan |
| CN105255964A (en) * | 2015-10-30 | 2016-01-20 | 宁波希诺亚海洋生物科技有限公司 | Production method of beta-glucan |
| CN115491311A (en) * | 2022-09-05 | 2022-12-20 | 武汉华美生物工程有限公司 | Method for crushing cell wall of pichia pastoris and application thereof |
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