Preparation method of yeast beta-glucan

Abstract

The invention discloses a yeast beta-glucan preparation method based on high pressure-microjet homogenization treatment, and belongs to the technical field of zymosan extraction. The method comprises the following steps: carrying out high pressure-microjet homogenization pretreatment on active dry yeast, then carrying out autolysis treatment, then extracting beta-glucan by an acid-alkali method, and finally carrying out freeze drying to obtain a high-purity product. The core of the method is that the yeast cell walls are subjected to physical wall breaking by adopting a high pressure-microjet homogenization technology, and the extraction rate and the purity of the beta-glucan are remarkably improved by combining with an optimized acid-alkali treatment process. Compared with a traditional enzymolysis method, the method has the advantages that damage of exogenous enzyme to the beta-glucan structure is avoided, the purity of the obtained product reaches 84% or above, the content of protein impurities is lower than 0.2%, a new way is provided for efficient utilization of yeast cell wall resources, and the method is suitable for the fields of food, medicine and health care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of yeast polysaccharide extraction. Specifically, it relates to a method for efficiently extracting high-purity β-glucan from yeast cell walls, and particularly to a preparation method for extracting β-glucan by homogenizing yeast using high-pressure microfluidization technology. Background Art

[0002] Yeast β-glucan is a natural polysaccharide from yeast cell walls, consisting of a β-(1,3)-D-glucan main chain and a β-(1,6)-D-glucan side chain. Research shows that yeast β-glucan has many useful biological activities. For example, it can activate the host's immune system, play an anti-tumor role through macrophages, natural killer cells and cytotoxic T cells, and play an anti-inflammatory role through immune-regulatory cells participating in innate immune responses, and inhibit cholesterol absorption as dietary fiber. Due to its non-toxicity and biodegradability, it has received great attention in the fields of food, cosmetics and biomedicine.

[0003] Currently, the extraction methods of yeast β-glucan mostly adopt the combination of acid-base method and enzymatic hydrolysis process (CN118948913A, CN110669680A, CN101570769A). However, the enzymatic hydrolysis method needs to introduce exogenous enzymes (such as alkaline protease, cellulase, pectinase, mannanase, β-glucanase), which easily leads to the degradation of the target product, thus reducing the product purity. In addition, the existing process has insufficient cell wall breaking efficiency, and the extraction rate is generally lower than 15%. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a preparation process of yeast β-glucan that combines high-pressure microfluidization homogenization pretreatment and optimized acid-base method to solve the problems of low product purity and low extraction rate in the existing process.

[0005] To achieve the above purpose, the inventors of the present invention conducted a large number of experimental studies and unremitting explorations, and finally obtained the following technical solution: A preparation method of yeast β-glucan, the method comprising the following steps:

[0006] (1) Suspend active dry yeast in phosphate buffer solution and perform high-pressure microfluidization homogenization treatment. The pressure is set as follows: high-pressure homogenization at 80 - 120 MPa, with 3 - 5 cycles of treatment; microfluidization homogenization at 40 - 60 MPa, with 3 - 5 cycles of treatment, to obtain a homogenized yeast suspension. High-pressure microfluidization physically breaks the yeast cell wall through shear force, cavitation effect and high-speed impact, releasing the internal β-glucan and avoiding the degradation of the polysaccharide structure by exogenous enzymes; multiple cycles of treatment ensure uniform cell wall breaking and improve the penetration efficiency of subsequent autolysis and acid-base treatment.

[0007] (2) Autolyze the homogenized yeast suspension under the following conditions: pH 4.5 - 6.5, temperature 45 - 55 °C, for 12 - 24 h. A mild acidic environment is conducive to activating endogenous yeast enzymes (such as proteases, phosphatases), decomposing cell contents (such as nucleic acids, proteins), and reducing impurity interference; the autolysis process retains the intact sugar chain structure of β-glucan, avoiding molecular breakage caused by drastic chemical treatment.

[0008] (3) After autolysis, inactivate the enzymes and perform centrifugal separation to obtain a muddy yeast cell wall precipitate. The preferred method for enzyme inactivation is high-temperature enzyme inactivation. High-temperature inactivation of endogenous enzymes can prevent the influence of residual enzymatic hydrolysis on product purity; centrifugal separation removes soluble impurities and enriches β-glucan in the cell wall residue; in addition, the supernatant after centrifugation generally needs to be recovered for the extraction of yeast cell contents.

[0009] (4) Treat the precipitate successively with alkali and acid. The alkali treatment step is as follows: add a sodium hydroxide solution with a volume 2 - 5 times that of the precipitate, and react at 60 - 70 °C for 1 - 3 h; the acid treatment step is as follows: after centrifugation, add an ice acetic acid solution with a volume 3 - 5 times that of the precipitate, and react at 70 - 80 °C for 1 - 2 h. The purpose of alkali treatment is to break the hydrogen bonds and hydrophobic interactions between β-glucan and mannan, protein, and dissolve non-target polysaccharides; the purpose of acid treatment is to degrade residual nucleic acids and cut the glycosidic bonds between β-glucan and impurities to further purify the target product; the optimization of temperature and duration aims to balance efficient extraction and structure protection, avoiding oxidation or hydrolysis of β-glucan caused by high-temperature alkali solution.

[0010] (5) After centrifugal washing, freeze-dry the precipitate to obtain yeast β-glucan powder. Freeze-drying removes moisture to obtain a loose powdery product, thereby improving the solubility and stability of yeast β-glucan.

[0011] Further preferably, in the preparation method of yeast β-glucan as described above, the pressure settings for high-pressure - microfluidic homogenization treatment in step (1) are as follows: high-pressure homogenization at 80 - 100 MPa, with 3 - 4 cycles; microfluidic homogenization at 45 - 55 MPa, with 3 - 4 cycles.

[0012] Further preferably, in the preparation method of yeast β-glucan as described above, the conditions for autolysis treatment in step (2) are as follows: pH 5.5 - 6.5, temperature 48 - 52 °C, time 12 - 20 h.

[0013] Further preferably, in the preparation method of yeast β-glucan as described above, the mass fraction of the sodium hydroxide solution in step (4) is 2.5% - 3.5%.

[0014] Further preferably, in the preparation method of yeast β-glucan as described above, the concentration of the glacial acetic acid solution in step (4) is 0.4 - 0.6 mol / L.

[0015] Further preferably, in the preparation method of yeast β-glucan as described above, the freeze-drying conditions in step (5) are: pre-freezing at -68 to -72 °C for 20 - 24 h, and then freeze-drying at -58 to -62 °C for 8 - 10 h.

[0016] Compared with the prior art, the preparation method of yeast β-glucan provided by the present invention has the following advantages and progressiveness:

[0017] (1) By replacing enzymatic hydrolysis treatment with physical cell wall breaking technology, the destruction of the polysaccharide structure is reduced. At the same time, the alkali treatment parameters are optimized, significantly increasing the extraction rate of yeast β-glucan to more than 12%, and the product purity reaches 84% - 88%.

[0018] (2) Compared with the traditional enzymatic hydrolysis method, the present invention avoids the destruction of the β-glucan structure by exogenous enzymes. The purity of the obtained product reaches more than 84%, and the protein impurity content is less than 0.2%, meeting the industry standards.

[0019] (3) The present invention is applicable to the efficient extraction of high-purity β-glucan from yeast cell walls, providing a new way for the efficient utilization of yeast cell wall resources, and is applicable to the fields of food, medicine and health products. Description of the Drawings

[0020] Figure 1 It is the absorbance scanning diagram of the sample of Example 1 at wavelengths of 400 - 700 nm.

[0021] Figure 2 It is the absorbance scanning diagram of the sample of Example 2 at wavelengths of 400 - 700 nm.

[0022] Figure 3 It is the absorbance scanning diagram of the sample of Example 3 at wavelengths of 400 - 700 nm.

[0023] Figure 4 It is the absorbance scanning diagram of the sample of Example 4 at wavelengths of 400 - 700 nm.

[0024] Figure 5 It is the standard curve diagram of the absorbance of yeast β-glucan standard solutions with different concentrations at a wavelength of 540 nm. Detailed Embodiments

[0025] The present invention will be further described in detail below through specific embodiments. For those steps or conditions of specific technical operations not specified in the embodiments, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0026] Example 1: Extraction of Yeast β-Glucan

[0027] Step 1, High-pressure Microfluidization Pretreatment: Activate the active dry yeast and suspend it in a pH 6.0 phosphate buffer solution, and then process it with a high-pressure microfluidization homogenizer. Perform high-pressure homogenization treatment at 90 MPa for 3 times and microfluidization homogenization treatment at 50 MPa for 3 times.

[0028] Step 2, Autolysis Treatment of the Homogenized Yeast Suspension: Autolyze at 50 °C and pH 6.0 for 18 h to activate the endogenous enzymes to decompose impurities.

[0029] Step 3, Enzyme Inactivation and Centrifugation: Inactivate the enzyme at 90 °C for 10 min and then centrifuge to collect the yeast cell wall precipitate.

[0030] Step 4, Acid-base Treatment: Add 4 times the volume of the precipitate of 3% NaOH solution and react at 60 °C for 2 h; after centrifugation, add 5 times the volume of 0.5 mol / L glacial acetic acid and react at 75 °C for 1 h.

[0031] Step 5, Freeze-drying: Centrifuge and wash the precipitate, place the precipitate in a freezer, pre-freeze at -70 °C for 22 h, then dry at -60 °C for 8 h, and obtain white β-glucan powder after grinding. The yield of β-glucan is 12.98%.

[0032] The purity of the β-glucan sample was quantitatively detected by the Congo red method, and the detection method is as follows:

[0033] First, make the absorbance standard curve of the yeast β-glucan standard solution at a wavelength of 540 nm: 1. Mix and dissolve the yeast β-glucan standard product with a 90% volume fraction of dimethyl sulfoxide solution in a 70 °C water bath, and prepare a yeast β-glucan standard solution with a concentration of 0 - 160 μg / mL. 2. Mix the solution with the same volume of Congo red dye solution with a concentration of 0.1 mg / mL and let it stand for 15 min. 3. Put the reacted sample into an ultraviolet spectrophotometer, scan and record its absorbance at a wavelength of 540 nm, and draw the absorbance standard curve, as Figure 5 shown.

[0034] Secondly, the β-glucan sample prepared in this example was fully mixed and dissolved with a dimethyl sulfoxide solution with a volume fraction of 90% in a 70°C water bath. The resulting solution was fully mixed with the same volume of Congo red dye solution with a concentration of 0.1 mg / mL and allowed to stand and react for 15 min. The reacted sample solution was placed in a UV spectrophotometer, scanned, and the absorbance scan of the sample at a wavelength of 400 - 700 nm was plotted, as shown in Figure 1 shown. By combining the absorbance scan results of this group of samples with the standard curve, it can be calculated that the purity of the β-glucan sample prepared in this example is 87.72%. This result shows that this method can effectively remove impurities such as proteins, chitosan, and chitin in the yeast cell wall, and the β-glucan prepared by this method has a high purity.

[0035] Example 2: Extraction of yeast β-glucan (more alkali solution used and shorter treatment time during alkali treatment)

[0036] Step 1, High-pressure microfluidization pretreatment: The active dry yeast was activated and suspended in a pH 6.0 phosphate buffer solution, and then treated with a high-pressure microfluidization homogenizer. It was homogenized at 90 MPa high pressure for 3 times and at 50 MPa microfluidization for 3 times.

[0037] Step 2, Autolysis of the homogenized yeast suspension: Autolysis was carried out at 50°C and pH 6.0 for 18 h to activate the endogenous enzymes to decompose impurities.

[0038] Step 3, Enzyme inactivation and centrifugation: After inactivating the enzyme at 90°C for 10 min, centrifugation was carried out to collect the yeast cell wall precipitate.

[0039] Step 4, Acid-base treatment: Add 5 times the precipitation volume of 3% NaOH solution and react at 70°C for 1 h; after centrifugation, add 5 times the volume of 0.5 mol / L glacial acetic acid and react at 75°C for 1 h.

[0040] Step 5, Freeze-drying: Centrifuge and wash the precipitate, place the precipitate in a freezer, pre-freeze at -70°C for 22 h, then dry at -60°C for 8 h, and obtain white β-glucan powder after grinding. The yield of β-glucan is 12.12%.

[0041] The β-glucan sample prepared in this example was fully mixed and dissolved with a dimethyl sulfoxide solution with a volume fraction of 90% in a 70°C water bath. The resulting solution was fully mixed with the same volume of Congo red dye solution with a concentration of 0.1 mg / mL and allowed to stand and react for 15 min. The reacted sample solution was placed in a UV spectrophotometer, scanned, and the absorbance scan of the sample at a wavelength of 400 - 700 nm was plotted, as shown in Figure 2As shown. By combining the absorbance scanning results of this group of samples with the standard curve, it can be calculated that the purity of the β-glucan sample prepared in this example is 84.46%. Compared with Example 1, the amount of lye used in this group of samples was increased, and the alkali treatment time was relatively reduced. Although the purity of the obtained sample decreased to a certain extent, it was still within a relatively high range.

[0042] Example 3: Extraction of Yeast β-Glucan (Less Lye Used during Alkali Treatment)

[0043] Step 1, High-pressure Microfluidization Pretreatment: Activate the active dry yeast and suspend it in a pH 6.0 phosphate buffer solution, and then treat it with a high-pressure microfluidization homogenizer. Perform high-pressure homogenization treatment at 90 MPa for 3 times and microfluidization homogenization treatment at 50 MPa for 3 times.

[0044] Step 2, Autolysis Treatment of the Homogenized Yeast Suspension: Autolyze at 50 °C and pH 6.0 for 18 h to activate endogenous enzymes to decompose impurities.

[0045] Step 3, Enzyme Inactivation and Centrifugation: Inactivate the enzyme at 90 °C for 10 min and then centrifuge to collect the yeast cell wall precipitate.

[0046] Step 4, Acid-base Treatment: Add 1 times the precipitation volume of 3% NaOH solution and react at 70 °C for 3 h; after centrifugation, add 5 times the volume of 0.5 mol / L glacial acetic acid and react at 75 °C for 1 h.

[0047] Step 5, Freeze-drying: Centrifuge and wash the precipitate, place the precipitate in a freezer, pre-freeze at -70 °C for 22 h, then dry at -60 °C for 8 h, and obtain white β-glucan powder after grinding. The yield of β-glucan is 8.03%.

[0048] Fully mix and dissolve the β-glucan sample prepared in this example with a 90% volume fraction of dimethyl sulfoxide solution in a 70 °C water bath. Fully mix the obtained solution with the same volume of Congo red dye solution with a concentration of 0.1 mg / mL and let it stand for reaction for 15 min. Put the reacted sample solution into a UV-visible spectrophotometer, scan and plot the absorbance scanning graph of the sample at a wavelength of 400 - 700 nm, as Figure 3 shown. By combining the absorbance scanning results of this group of samples with the standard curve, it can be calculated that the purity of the β-glucan sample prepared in this example is 80.40%. The amount of lye used in this group of samples was reduced, and the yield of β-glucan was significantly lower than that of Example 1 and Example 2, and the purity of the glucan was also lower.

[0049] Example 4: Extraction of Yeast β-Glucan (Enzymatic Cell Wall Disruption)

[0050] Step 1, Yeast cell wall lysing enzyme: Activate the active dry yeast and suspend it in a pH 6.0 phosphate buffer solution, and add 1.2% of the yeast cell wall lysing enzyme (Nanning Pangbo Bioengineering Co., Ltd.) based on the dry weight of the yeast.

[0051] Step 2, Autolysis treatment of the homogenized yeast suspension: Autolyze at 50 °C and pH 4.5 for 18 h to activate the endogenous enzymes to decompose impurities.

[0052] Step 3, Enzyme inactivation and centrifugation: Inactivate the enzyme at 90 °C for 10 min and then centrifuge to collect the yeast cell wall precipitate.

[0053] Step 4, Acid-base treatment: Add 3% NaOH solution with a volume 4 times that of the precipitate, react at 60 °C for 2 h; after centrifugation, add 0.5 mol / L glacial acetic acid with a volume 5 times that of the precipitate and react at 75 °C for 1 h.

[0054] Step 5, Freeze-drying: Centrifuge and wash the precipitate, place the precipitate in a freezer, pre-freeze at -70 °C for 22 h, then dry at -60 °C for 8 h, and obtain white β-glucan powder after grinding. The yield of β-glucan is 9.41%.

[0055] Fully mix and dissolve the β-glucan sample prepared in this example with a 90% volume fraction of dimethyl sulfoxide solution in a 70 °C water bath. Mix the resulting solution with the same volume of Congo red dye solution with a concentration of 0.1 mg / mL and let it stand for reaction for 15 min. Put the reacted sample solution into an ultraviolet spectrophotometer, scan and plot the absorbance scan graph of the sample at a wavelength of 400 - 700 nm, as Figure 4 shown. Combining the absorbance scan results of this group of samples with the standard curve calculation, it can be known that the purity of the β-glucan in the sample prepared in this example is 74.25%. This group uses yeast cell wall lysing enzyme to treat yeast cells, and under suitable conditions, β-glucan can be decomposed into oligosaccharides such as cellobiose and maltose. Therefore, its β-glucan yield and glucan purity are much lower than those in Example 1 and Example 2. This result shows that compared with the traditional enzymatic cell wall breaking method, the homogenization treatment method using high-pressure microfluidics technology in the present invention has significant advantages in improving the yield and purity of yeast β-glucan.

[0056] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. A preparation method of yeast β-glucan, characterized in that, The method comprises the following steps: (1) Suspend active dry yeast in phosphate buffer solution and perform high-pressure - microfluidic jet homogenization treatment. The pressure is set as follows: high-pressure homogenization at 80 - 120 MPa, with cyclic treatment for 3 - 5 times; microfluidic jet homogenization at 40 - 60 MPa, with cyclic treatment for 3 - 5 times, to obtain a homogenized yeast suspension; (2) Perform autolysis treatment on the homogenized yeast suspension. The treatment conditions are: pH 4.5 - 6.5, temperature 45 - 55 °C, lasting for 12 - 24 h; (3) Inactivate enzymes after autolysis ends, and perform centrifugal separation to obtain a muddy yeast cell wall precipitate; (4) Treat the precipitate successively with alkali and acid. The alkali treatment step is: add a sodium hydroxide solution with a volume 2 - 5 times that of the precipitate, and react at 60 - 70 °C for 1 - 3 h; the acid treatment step is: after centrifugation, add an acetic acid solution with a volume 3 - 5 times that of the precipitate, and react at 70 - 80 °C for 1 - 2 h; (5) After centrifugal washing, freeze-dry the precipitate to obtain yeast β-glucan powder.

2. The preparation method of yeast β-glucan according to claim 1, wherein: In step (1), the pressure setting for high-pressure - microfluidic jet homogenization treatment is: high-pressure homogenization at 80 - 100 MPa, with cyclic treatment for 3 - 4 times; microfluidic jet homogenization at 45 - 55 MPa, with cyclic treatment for 3 - 4 times.

3. The preparation method of yeast β-glucan according to claim 1, wherein: In step (2), the autolysis treatment conditions are: pH 5.5 - 6.5, temperature 48 - 52 °C, and time 12 - 20 h.

4. The preparation method of yeast β-glucan according to claim 1, characterized in that: In step (4), the mass fraction of the sodium hydroxide solution is 2.5% - 3.5%.

5. The preparation method of yeast β-glucan according to claim 1, wherein: In step (4), the concentration of the acetic acid solution is 0.4 - 0.6 mol / L.

6. The preparation method of yeast β-glucan according to claim 1, wherein: In step (5), the freeze-drying conditions are: pre-freezing at -68 - -72 °C for 20 - 24 h, and then freeze-drying at -58 - -62 °C for 8 - 10 h.

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