A method for determining the content of yeast β-glucan in yeast culture

Abstract

The present invention discloses a method for determining the content of yeast β-glucan in yeast culture, which belongs to the field of chemical analysis technology. The method includes: pretreatment; total glucan hydrolysis; α-glucan hydrolysis; cereal β-glucan hydrolysis; derivatization reaction; drawing and determination of standard curve. The method of the present invention has high precision, good repeatability and feasibility, and has reference value for the accurate detection of the content of yeast β-glucan in yeast culture. The method of the present invention will provide a reference for the further development and preparation of yeast culture, and provide more valuable scientific basis for the development of related fields. Through this method, we can more accurately evaluate the content of yeast β-glucan in yeast culture, thereby ensuring the accuracy of product quality.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and more particularly to a method for determining the content of yeast beta-glucan in yeast culture. Background Art

[0002] In recent years, yeast culture (YC) has played an important role in animal husbandry as a green and efficient alternative to antibiotics. YC can optimize the nutritional value of feed and actively regulate the immune system of animals, thereby promoting the growth of animals and enhancing their production efficiency. Studies have shown that yeast β-glucan in YC is one of its main active ingredients. It is not only an efficient immune activator, but also has anti-tumor, antioxidant, anti-inflammatory effects, promotes wound healing, and enhances intestinal immune function.

[0003] Glucan is the most abundant natural polysaccharide among all carbohydrate polymers. Based on the different structures, glucan is divided into α-glucan and β-glucan. α-glucan is stored in plants and animals in the form of starch, sucrose, animal glycogen, etc., and is mainly used as an energy source. β-glucan is naturally present in grains, bacteria, mushrooms, yeast and other substances, and its physical and chemical properties vary significantly depending on the source. Studies have shown that grain β-glucan (such as wheat, corn and beans) is a linear homogeneous polysaccharide composed of glucose linked by continuous β-1,4 and intermittent β-1,3 glycosidic bonds, which mainly affects animal metabolism, such as lowering cholesterol, and has potential benefits for cardiovascular health; while yeast β-glucan is a highly branched macromolecular polysaccharide, whose main chain is composed of D-glucose linked by β-1,3 glycosidic bonds, and the side chains are composed of D-glucose linked by β-1,6 glycosidic bonds, which mainly affects the immune system and has anti-inflammatory, anti-cancer and anti-infective properties. In YC, the above α-glucan and β-glucan exist at the same time, but yeast β-glucan itself is difficult to be directly measured. Therefore, the accurate determination of yeast β-glucan with immune activity in YC has always been a difficulty in research.

[0004] At present, there is a lack of standard detection methods for accurately determining the content of yeast β-glucan in YC both at home and abroad. Therefore, in order to evaluate the quality of products containing yeast β-glucan, it is important to quantitatively analyze the content of yeast β-glucan in YC, improve various detection indicators, and develop a method for accurately determining the content of yeast β-glucan in YC. The reported detection methods of yeast β-glucan mainly include enzymatic hydrolysis, high performance liquid chromatography (HPLC), nuclear magnetic resonance, etc. HPLC is based on strong acid hydrolysis of glucan into glucose, and the glucose content is converted into glucan content. However, other glucose polymers present in the sample matrix may also release glucose, which will lead to inaccurate measurement and the content may be too high. The nuclear magnetic resonance method is to dissolve glucan in DMSO and measure the internal standard by 1HNMR spectroscopy to obtain the β-glucan content. This method requires that the sample is easily soluble in DMSO and does not contain any interfering substances. Enzymatic hydrolysis has become an important technology for analyzing yeast β-glucan due to its high specificity and accuracy. This technology was originally proposed by McCleary, and after subsequent improvements, it was developed into a kit form and became a commercial classic detection method. The enzymatic hydrolysis method has been recognized by several international authoritative organizations, including the American Association of Analytical Chemists (AOAC International, 995.16). Nevertheless, this method is not suitable for the analysis of yeast β-glucan in the presence of other β-glucans, such as cereal β-glucan. In addition, the required enzymes are usually expensive, and these factors have limited its application in the determination of yeast β-glucan in YC to a certain extent.

[0005] Therefore, how to develop a method for determining the content of yeast β-glucan in yeast culture is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides a method for determining the content of yeast β-glucan in yeast culture.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for determining the content of yeast β-glucan in a yeast culture comprises the following steps:

[0009] (1) Pretreatment: The yeast culture sample to be tested, the yeast β-glucan reference substance of known purity, and the grain β-glucan reference substance of known purity are crushed and sieved for later use;

[0010] (2) Total glucan hydrolysis: Weigh the pretreated sample to be tested and a yeast β-glucan reference substance of known purity, add sulfuric acid aqueous solution to each of them, place in an ice water bath, then add ultrapure water to place in a boiling water bath, transfer the solution after the boiling water bath to a volumetric flask with sodium acetate buffer, then add sodium hydroxide aqueous solution to the volumetric flask for neutralization, finally make up to volume with sodium acetate buffer, centrifuge the hydrolyzed solution, collect the supernatant into a centrifuge tube, add a suspension of exo-1,3-β-glucanase and β-glucosidase to the centrifuge tube, and heat in a water bath to obtain sample solution A and reference substance solution A;

[0011] α-glucan hydrolysis: weigh the pretreated sample to be tested and the yeast β-glucan reference substance of known purity, add sodium hydroxide aqueous solution to each of them, place in an ice water bath, and stir magnetically, then add sodium acetate buffer to mix, then add a suspension of amyloglucosidase and sucrase and a suspension of trehalase to heat in a water bath, finally transfer the solution after water bath heating to a volumetric flask with ultrapure water and make up to volume, centrifuge the hydrolyzed solution, collect the supernatant into a centrifuge tube, and obtain sample solution B and reference substance solution B;

[0012] Hydrolysis of cereal β-glucan: Weigh the pretreated sample to be tested and the cereal β-glucan reference substance of known purity, add ethanol aqueous solution and sodium phosphate buffer respectively, place in a boiling water bath, then heat in a water bath, add the suspension of endo-1,3-1,4-β-glucan hydrolase, continue to heat in a water bath, finally transfer the solution heated in a water bath to a volumetric flask with sodium acetate buffer and make up to volume, centrifuge the hydrolyzed solution, collect the supernatant into a centrifuge tube, add the suspension of β-glucosidase into the centrifuge tube, and heat in a water bath to obtain sample solution C and reference substance solution C;

[0013] (3) Derivatization reaction: take the sample solution A, sample solution B, sample solution C, reference solution A, reference solution B, and reference solution C obtained in step (2), add PMP-methanol solution and sodium hydroxide aqueous solution respectively, heat in a water bath, then add hydrochloric acid, and extract repeatedly with chloroform for multiple times, finally take the upper aqueous phase for centrifugation, collect the supernatant and filter, and obtain test solution A, test solution B, test solution C, test solution A control, test solution B control, and test solution C control;

[0014] (4) Drawing and determination of standard curve:

[0015] Dissolve the glucose standard in ultrapure water to prepare a 1 mg / mL standard stock solution, store at 4°C, dilute the standard stock solution into a series of standard working solutions with concentrations of 0.5, 5, 10, 50, 100, and 400 μg / mL, and prepare them before use;

[0016] The standard series working solutions are subjected to derivatization reaction according to step (3);

[0017] The standard series working solutions after the derivatization reaction and the test solution A, test solution B, test solution C, test solution A control, test solution B control and test solution C control were measured by high performance liquid chromatography, and the high performance liquid chromatography conditions were:

[0018] Column: Acclaim TM C30, 5 μm 4.6 × 250 mm HPLC column;

[0019] Mobile phase: a mixed solution of 0.1 mol / L ammonium acetate aqueous solution with a pH of 5.5 and acetonitrile in a volume ratio of 75:25;

[0020] Flow rate: 1.0 mL / min;

[0021] Column temperature: 35°C;

[0022] Detector: UV detector;

[0023] Detection wavelength: 254nm;

[0024] Injection volume: 10 μL;

[0025] The target glucose peak of the standard series working solution was analyzed to obtain the chromatographic peak area. The standard curve was drawn with the concentration of the standard series working solution as the horizontal axis and the chromatographic peak area of ​​the standard series working solution as the vertical axis to obtain the linear regression equation. The target glucose peaks of the test solution A, the test solution B, the test solution C, the test solution A control, the test solution B control and the test solution C control were analyzed to obtain the chromatographic peak areas. The concentrations corresponding to the chromatographic peak areas of the test solution A, the test solution B, the test solution C, the test solution A control, the test solution B control and the test solution C control were calculated by the linear regression equation. The content of yeast β-glucan in the yeast culture sample to be tested was calculated according to formulas (1)-(11). The calculation formula is:

[0026] X D =X A -X B -X C (1)

[0027]

[0028] X 酵母对照-检测值 =X A对照 -X B对照 (7)

[0029] X 谷物对照-检测值 =Xc对照 (8)

[0030]

[0031] Where:

[0032] X D -The content of yeast β-glucan in the yeast culture sample to be tested, expressed as mass percentage, %;

[0033] X A -The content of total glucan in the test solution A, expressed as mass percentage, %;

[0034] X B- The content of α-glucan in the test solution B is expressed as mass percentage, %;

[0035] X C- The content of cereal β-glucan in the test solution C is expressed as mass percentage, %;

[0036] X A对照 -The total glucan content in the test solution A control, expressed as mass percentage, %;

[0037] X B对照 -The content of α-glucan in the test solution B control, expressed as mass percentage, %;

[0038] X 酵母对照-检测值 -The total glucan content in the test solution A control minus the α-glucan content in the test solution B control, that is, the yeast β-glucan content detected in the test solution A control and the test solution B control, expressed as mass percentage, %;

[0039] X 谷物对照-检测值 - The content of cereal β-glucan in the test solution C control, that is, the content of cereal β-glucan detected in the test solution C control, expressed as mass percentage, %;

[0040] X C对照 -X 谷物对照-检测值 , the content of cereal β-glucan in the test solution C control, that is, expressed as mass percentage, %;

[0041] c A -Concentration corresponding to the chromatographic peak area of ​​glucose in test solution A, μg / mL;

[0042] c B -The concentration corresponding to the chromatographic peak area of ​​glucose in the test solution B, μg / mL;

[0043] c C-Concentration corresponding to the chromatographic peak area of ​​glucose in the test solution C, μg / mL;

[0044] c A对照 -Concentration of glucose chromatographic peak area corresponding to the test solution A control, μg / mL;

[0045] c B对照 -Concentration corresponding to the chromatographic peak area of ​​glucose in the test solution B control, μg / mL;

[0046] c C对照 -Concentration corresponding to the chromatographic peak area of ​​glucose in the test solution C control, μg / mL;

[0047] m A - Total glucan hydrolysis Weigh the mass of the sample to be tested, g;

[0048] m B -α-glucan hydrolysis Weigh the mass of the sample to be tested, g;

[0049] m C - Cereal β-glucan hydrolysis Weigh the mass of the sample to be tested, g;

[0050] m A对照 -Total glucan hydrolysis Weigh the mass of yeast β-glucan reference substance, g;

[0051] m B对照 -α-glucan hydrolysis Weigh the mass of yeast β-glucan reference substance, g;

[0052] m C对照 - Cereal β-glucan hydrolysis Weigh the mass of cereal β-glucan reference substance, g;

[0053] V A -Total glucan hydrolysis sample volume, mL;

[0054] V B -The fixed volume of the α-glucan hydrolyzed sample, mL;

[0055] V C - Volume of the sample to be tested for the hydrolysis of cereal β-glucan, mL;

[0056] V A对照 -Total glucan hydrolyzed yeast β-glucan reference volume, mL;

[0057] V B对照 -α-glucan hydrolyzed yeast β-glucan reference standard volume, mL;

[0058] V C对照 - Cereal β-glucan hydrolyzed cereal β-glucan reference standard volume, mL;

[0059] F 酵母 - Correction factor for low results caused by the destruction or loss of total glucan and α-glucan in the test sample during the test;

[0060] F 谷物 - Correction factor for low results caused by destruction or loss of cereal β-glucan in the test sample during the test;

[0061] P 酵母 -Purity of yeast β-glucan reference substance, %;

[0062] P 谷物 -Purity of cereal β-glucan reference substance, %;

[0063] W 酵母 -Moisture content of yeast β-glucan reference, %;

[0064] W 谷物 - moisture content of the cereal β-glucan reference, %;

[0065] 8.5-The volume dilution factor of the supernatant collected from the hydrolysis of total glucan or cereal β-glucan to prepare the sample solution or reference solution, and then add PMP-methanol solution, sodium hydroxide aqueous solution and hydrochloric acid;

[0066] 4.25-α-glucan hydrolysis, collect the supernatant to prepare the sample solution or reference solution, and then add PMP-methanol solution, sodium hydroxide aqueous solution and hydrochloric acid to dilute the supernatant by volume;

[0067] 0.9 - coefficient for converting glucose content into glucan content;

[0068] X A对照 , X B对照 , X C对照 and X D Perform multiple repeated measurements, and express the measurement results as the arithmetic mean ± standard deviation of the multiple repeated measurement results, retaining 3 significant figures.

[0069] Furthermore, in step (1), the yeast culture to be tested is sampled by the five-point method, and the yeast culture sample to be tested, the yeast β-glucan reference substance of known purity, and the grain β-glucan reference substance of known purity are crushed and passed through a 60-mesh sieve, and stored at -20°C for later use.

[0070] Further, in step (2), the total glucan is hydrolyzed by weighing 0.0500-0.1200 g of the pretreated sample to be tested and 0.0500-0.1200 g of a yeast β-glucan reference substance of known purity, adding 2.0 mL of a 12 mol / L sulfuric acid aqueous solution to each of them, and then placing them in an ice water bath for 2 h, then adding 10 mL of ultrapure water and placing them in a boiling water bath for 2 h, and then transferring the solution after the boiling water bath to a 100 mL volumetric flask with a pH 4.5, 200 mmol / L sodium acetate buffer, and then adding 6 mL of an 8 mol / L aqueous solution to the volumetric flask. / L sodium hydroxide aqueous solution was used for neutralization, and finally the volume was made up to 100mL with pH4.5 and 200mmol / L sodium acetate buffer. 1-2mL of the hydrolyzed solution was centrifuged at a speed of 13000r / min for 5min, and 0.2mL of the supernatant was collected into a 15mL centrifuge tube. 0.2mL of a suspension of exo-1,3-β-glucanase and β-glucosidase was added to the centrifuge tube, and then heated in a water bath at 40°C for 60min to obtain 400μL of sample solution A and 400μL of reference solution A, respectively.

[0071] Further, in step (2), α-glucan hydrolysis: weigh 0.0500-0.1200g of the pretreated sample to be tested and 0.0500-0.1200g of yeast β-glucan reference substance of known purity, add a magnetic stirring bar with a diameter of 5mm and a length of 15mm, add 2mL of 1.7mol / L sodium hydroxide aqueous solution and ice water bath, and magnetically stir for 20min while ice water bath, the stirring speed is 600-1000r / min, and after the stirring is completed, add 8mL The pH value is 3.8, and the concentration is 1.2 mol / L sodium acetate buffer for mixing. Then, 0.4 mL of amyloglucosidase and sucrase suspension and 0.1 mL of trehalase suspension are added and heated in a 40°C water bath for 60 min. Finally, ultrapure water is used to transfer the solution heated in a water bath to a 100 mL volumetric flask and make up the volume. 2-4 mL of the hydrolyzed solution is centrifuged at a speed of 13000 r / min for 5 min. 0.4 mL of the supernatant is collected in a 15 mL centrifuge tube to obtain 400 μL of sample solution B and 400 μL of reference solution B, respectively.

[0072] Further, in step (2), the cereal β-glucan is hydrolyzed by weighing 0.0500-0.1200 g of the pretreated sample to be tested and 0.0500-0.1200 g of a cereal β-glucan reference substance of known purity, and adding 0.2 mL of a 50% ethanol aqueous solution and 4 mL of The mixture was boiled in a water bath for 5 min with a pH value of 6.5 and a concentration of 20 mmol / L sodium phosphate buffer, then heated in a water bath at 50°C for 5 min, and then 0.2 mL of a suspension of endo-1,3-1,4-β-glucan hydrolase was added, and the mixture was heated in a water bath at 50°C for 60 min. Finally, the solution heated in a water bath was transferred to a 10-50 mL centrifuge tube with a pH value of 4.0 and a concentration of 200 mmol / L sodium acetate buffer and fixed to 10-50 mL. 2-4 mL of the hydrolyzed solution was centrifuged at a speed of 13000 r / min for 5 min. 0.2 mL of the supernatant was collected and transferred to a 15 mL centrifuge tube. 0.2 mL of a suspension of β-glucosidase was added to the centrifuge tube and heated in a water bath at 50°C for 10 min to obtain 400 μL of a sample solution C and 400 μL of a reference solution C, respectively.

[0073] Further, in step (3), 400 μL of each of the sample solution A, sample solution B, sample solution C, reference solution A, reference solution B, and reference solution C obtained in step (2) are taken, 400 μL of PMP-methanol solution with a PMP concentration of 0.5 mol / L and 400 μL of sodium hydroxide aqueous solution with a concentration of 0.3 mol / L are added, respectively, and then heated in a water bath at 70°C for 60 min, and then 500 μL of 0.3 mol / L hydrochloric acid is added, and then the extraction is repeated 4 times with 3 mL of chloroform each time, and finally the upper aqueous phase is taken for centrifugation at a centrifugal speed of 10000 r / min and a centrifugal time of 2 min. The supernatant is collected and filtered through a 0.22 μm syringe filter to obtain test solution A, test solution B, test solution C, test solution A control, test solution B control, and test solution C control.

[0074] Further, the preparation method of the suspension of exo-1,3-β-glucanase and β-glucosidase comprises the following steps:

[0075] To 2.0 mL of a suspension having an enzyme concentration of 100 U / mL of exo-1,3-β-glucanase and an enzyme concentration of 20 U / mL of β-glucosidase, 9 mL of a sodium acetate buffer having a pH of 4.5 and a concentration of 200 mmol / L was added to dilute the suspension to 11 mL, and the suspension was divided into equal portions for storage;

[0076] The suspension of amyloglucosidase and sucrase is:

[0077] A suspension of amyloglucosidase and sucrase in a 50% glycerol aqueous solution as solvent (20 mL in total), wherein the enzyme activity of amyloglucosidase is 1630 U / mL and the enzyme activity of sucrase is 500 U / mL, is used directly;

[0078] Trehalase suspension is:

[0079] Trehalase suspension, 5 mL, used directly;

[0080] The preparation method of the suspension of endo-1,3-1,4-β-glucanohydrolase comprises the following steps:

[0081] Dilute 1.0 mL of the initial suspension of endo-1,3-1,4-β-glucanohydrolase to 20.0 mL with a sodium phosphate buffer having a pH of 6.5 and a concentration of 20 mmol / L, so that the final enzyme concentration of the endo-1,3-1,4-β-glucanohydrolase is 50 U / mL, and store the suspension in equal portions;

[0082] The preparation method of the suspension of β-glucosidase comprises the following steps:

[0083] 1.0 mL of the initial suspension of β-glucosidase was diluted to 20.0 mL with a pH 4.0, 50 mmol / L sodium acetate buffer to make the final enzyme concentration of β-glucosidase 2 U / mL, and the suspension was stored in equal portions.

[0084] Further, in step (2), the preparation method of the sodium acetate buffer having a pH of 4.5 and a concentration of 200 mmol / L comprises the following steps:

[0085] Add 5.8 mL of glacial acetic acid to 450 mL of ultrapure water, adjust the pH to 4.5 by adding 1 mol / L sodium hydroxide solution, and make up to 500 mL with ultrapure water;

[0086] The preparation method of the sodium acetate buffer solution with a pH of 3.8 and a concentration of 1.2 mol / L comprises the following steps:

[0087] Add 34.3 mL of glacial acetic acid to 400 mL of distilled water, adjust the pH to 3.8 with 4 mol / L sodium hydroxide, and make up to 500 mL with ultrapure water;

[0088] The preparation method of a sodium acetate buffer having a pH of 4.0 and a concentration of 200 mmol / L comprises the following steps:

[0089] Add 5.8 mL of glacial acetic acid to 450 mL of ultrapure water, adjust the pH to 4.0 by adding 1 mol / L sodium hydroxide solution, and make up to 500 mL with ultrapure water;

[0090] The preparation method of the sodium phosphate buffer solution with a pH of 6.5 and a concentration of 20 mmol / L comprises the following steps:

[0091] Dissolve 1.56 g of sodium dihydrogen phosphate dihydrate in 450 mL of ultrapure water, adjust the pH to 6.5 by adding 100 mmol / L sodium hydroxide solution, and make up to 500 mL with ultrapure water;

[0092] The preparation method of the sodium acetate buffer solution with a pH of 4.0 and a concentration of 50 mmol / L comprises the following steps:

[0093] 1.45 mL of glacial acetic acid was added to 450 mL of ultrapure water, the pH value was adjusted to 4.0 by adding 1 mol / L sodium hydroxide solution, and the volume was fixed to 500 mL.

[0094] Beneficial effects of the present invention: The method of the present invention has high precision, good repeatability and feasibility, and has reference value for the accurate detection of the yeast β-glucan content in yeast culture. The method of the present invention will provide a reference for the further development and preparation of yeast culture, and provide more valuable scientific basis for the development of related fields. Through this method, we can more accurately evaluate the content of yeast β-glucan in yeast culture, thereby ensuring the accuracy of product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 The effect of the amount and ratio of enzyme B1 and enzyme B2 on the glucose concentration in the sample;

[0096] Figure 2 The results of the effect of derivatization reaction time on the concentration of glucose derivatives in samples;

[0097] Figure 3 Results of the effect of extraction times on PMP peak area;

[0098] Figure 4 In the figure, Figure a shows the separation effect of the C18 column, and Figure b shows the separation effect of the C30 column;

[0099] Figure 5 In the figure, Figure a is the chromatogram of glucose PMP derivatives in glucose standards and test group samples, and Figure b is the glucose standard curve. DETAILED DESCRIPTION

[0100] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0101] 1. Instruments and equipment

[0102] Agilent 1260 InfinityⅡ liquid chromatography system (Agilent, USA); chromatographic column Acclaim C30 (4.6 mm×250 mm, 5 μm) (Thermo Fisher Scientific, USA); Milli-Q plus ultrapure water system (Millipore, USA); magnetic stirrer (Thermo Fisher Scientific, USA); vortex mixer (IKA, Germany);

[0103] 2. Reagents and raw materials

[0104] Glucose standard, 1-phenyl-3-methyl-5-pyrazolone (PMP) (analytical grade, Shanghai Aladdin Company); yeast β-glucan reference substance (β-glucan reference substance derived from yeast, P in the calculation formula 酵母 (100%-W 酵母 ) value was 47%, hereinafter referred to as yeast control, purchased from ), Cereal β-glucan reference substance (β-glucan reference substance derived from cereals, the calculation formula P 谷物 (100%-W 谷物 ) was 4.1%, hereinafter referred to as the grain reference, purchased from ).

[0105] A1 enzyme: prepare a suspension of exo-1,3-β-glucanase and β-glucosidase: add 9 mL of a pH 4.5, 200 mmol / L sodium acetate buffer to 2.0 mL of a suspension of exo-1,3-β-glucanase at an enzyme concentration of 100 U / mL and β-glucosidase at an enzyme concentration of 20 U / mL, dilute to 11 mL, and store the suspension in equal portions;

[0106] B1 enzyme: 20 mL of a suspension of amyloglucosidase and sucrase in a 50% glycerol aqueous solution as solvent, wherein the enzyme activity of amyloglucosidase is 1630 U / mL and the enzyme activity of sucrase is 500 U / mL, which can be used directly:

[0107] B2 enzyme: Trehalase suspension, 5 mL, used directly, purchased from MK-YBGL

[0108] Enzyme C1: Prepare a suspension of endo-1,3-1,4-β-glucanohydrolase: dilute 1.0 mL of the initial suspension of endo-1,3-1,4-β-glucanohydrolase to 20.0 mL with a pH 6.5, 20 mmol / L sodium phosphate buffer to a final enzyme concentration of 50 U / mL of endo-1,3-1,4-β-glucanohydrolase, and store the suspension in equal portions;

[0109] C2 enzyme: Prepare a suspension of β-glucosidase: dilute 1.0 mL of the initial suspension of β-glucosidase to 20.0 mL with a pH 4.0, 50 mmol / L sodium acetate buffer to make the final enzyme concentration of β-glucosidase 2 U / mL, and store the suspension in equal portions.

[0110] The preparation method of a sodium acetate buffer having a pH of 4.5 and a concentration of 200 mmol / L comprises the following steps:

[0111] Add 5.8 mL of glacial acetic acid to 450 mL of ultrapure water, adjust the pH to 4.5 by adding 1 mol / L sodium hydroxide solution, and make up to 500 mL with ultrapure water;

[0112] The preparation method of the sodium acetate buffer solution with a pH of 3.8 and a concentration of 1.2 mol / L comprises the following steps:

[0113] Add 34.3 mL of glacial acetic acid to 400 mL of distilled water, adjust the pH to 3.8 with 4 mol / L sodium hydroxide, and make up to 500 mL with ultrapure water;

[0114] The preparation method of a sodium acetate buffer having a pH of 4.0 and a concentration of 200 mmol / L comprises the following steps:

[0115] Add 5.8 mL of glacial acetic acid to 450 mL of ultrapure water, adjust the pH to 4.0 by adding 1 mol / L sodium hydroxide solution, and make up to 500 mL with ultrapure water;

[0116] The preparation method of the sodium phosphate buffer solution with a pH of 6.5 and a concentration of 20 mmol / L comprises the following steps:

[0117] Dissolve 1.56 g of sodium dihydrogen phosphate dihydrate in 450 mL of ultrapure water, adjust the pH to 6.5 by adding 100 mmol / L sodium hydroxide solution, and make up to 500 mL with ultrapure water;

[0118] The preparation method of the sodium acetate buffer solution with a pH of 4.0 and a concentration of 50 mmol / L comprises the following steps:

[0119] 1.45 mL of glacial acetic acid was added to 450 mL of ultrapure water, the pH value was adjusted to 4.0 by adding 1 mol / L sodium hydroxide solution, and the volume was fixed to 500 mL.

[0120] Example 1

[0121] The method for determining the content of yeast β-glucan in yeast culture comprises the following steps:

[0122] (1) The yeast culture to be tested is sampled by the five-point method, and the yeast culture sample to be tested, the yeast β-glucan reference substance of known purity, and the grain β-glucan reference substance of known purity are crushed and passed through a 60-mesh sieve, and stored at -20°C for later use.

[0123] (2) Total glucan hydrolysis: Weigh about 0.1000 g of the pretreated sample and about 0.1000 g of yeast β-glucan reference substance of known purity, add 2.0 mL of 12 mol / L sulfuric acid aqueous solution to each of them, and place in an ice water bath for 2 h. Then, add 5 mL of ultrapure water and vortex to mix. The power of the vortex oscillation is 50 W, the speed is 1800 r / min, and the oscillation mode is touched. The vortex oscillation time is 1 min. Then, add 5 mL of ultrapure water and place in a boiling water bath for 2 h. Then, transfer the solution after the boiling water bath to a 100 mL volumetric flask with a sodium acetate buffer solution of pH 4.5 and a concentration of 200 mmol / L. Then, 6 mL of 8 mol / L sodium hydroxide aqueous solution was added to the volumetric flask for neutralization. Finally, the volume was made up to 100 mL with a pH 4.5, 200 mmol / L sodium acetate buffer. 2 mL of the hydrolyzed solution was centrifuged at a speed of 13000 r / min for 5 min. 0.2 mL of the supernatant was collected into a 15 mL centrifuge tube. 0.2 mL of a suspension of exo-1,3-β-glucanase and β-glucosidase was added to the centrifuge tube, and then heated in a water bath at 40°C for 60 min to obtain 400 μL of sample solution A and 400 μL of reference solution A, respectively.

[0124] α-Glucan hydrolysis: weigh about 0.0600g of the pretreated sample and about 0.1000g of yeast β-glucan reference substance of known purity, add a magnetic stirrer with a diameter of 5mm and a length of 15mm, add 2mL of 1.7mol / L sodium hydroxide aqueous solution and ice water bath, and magnetically stir for 20min at the same time, with a stirring speed of 800r / min. After stirring, add 8mL The pH value is 3.8, and the concentration is 1.2 mol / L sodium acetate buffer for mixing. Then, 0.4 mL of amyloglucosidase and sucrase suspension and 0.1 mL of trehalase suspension are added and heated in a 40°C water bath for 60 min. Finally, the solution heated in a water bath is transferred to a 100 mL volumetric flask with ultrapure water and the volume is made up to 100 mL with ultrapure water. 2 mL of the hydrolyzed solution is centrifuged at a speed of 13000 r / min for 5 min. 0.4 mL of the supernatant is collected in a 15 mL centrifuge tube to obtain 400 μL of sample solution B and 400 μL of reference solution B, respectively.

[0125] Hydrolysis of cereal β-glucan: weigh about 0.1000g of the pretreated sample to be tested and about 0.1000g of a cereal β-glucan reference substance of known purity, add 0.2mL of 50% ethanol aqueous solution and 4mL of pH 6.5, 20mmol / L sodium phosphate buffer, respectively, and boil in a water bath for 5min, then heat in a 50℃ water bath for 5min, then add 0.2mL of endo-1,3-1,4-β-glucan hydrolase suspension, continue to heat in a 50℃ water bath for 60min, and finally transfer the solution heated in a water bath to a 50mL centrifuge tube with pH 4.0, 200mmol / L sodium acetate buffer, and precipitate in a 50mL centrifuge tube with pH 4.0, 200mmol / L sodium acetate buffer. The sample solution to be tested was diluted to 50 mL, and the cereal β-glucan reference solution was diluted to 30 mL. After the volume was diluted, 2 mL of the hydrolyzed solution was centrifuged at a speed of 13000 r / min for 5 min. 0.2 mL of the supernatant was collected and transferred to a 15 mL centrifuge tube. 0.2 mL of β-glucosidase suspension was added to the centrifuge tube, and the tube was heated in a 50°C water bath for 10 min to obtain 400 μL of sample solution C and 400 μL of reference solution C, respectively.

[0126] (3) Take the sample solution A, sample solution B, sample solution C, reference solution A, reference solution B and reference solution C obtained in step (2), add 400 μL of PMP-methanol solution with a PMP concentration of 0.5 mol / L and 400 μL of sodium hydroxide aqueous solution with a concentration of 0.3 mol / L, respectively, and heat in a water bath at 70°C for 60 min, then add 500 μL of 0.3 mol / L hydrochloric acid, and then repeat the extraction with 3 mL of chloroform four times each time, finally take the upper aqueous phase and centrifuge it at a centrifugal speed of 10000 r / min for 2 min, collect the supernatant and filter it with a 0.22 μm syringe filter to obtain test solution A, test solution B, test solution C, test solution A control, test solution B control and test solution C control.

[0127] (4) Drawing and determination of standard curve:

[0128] Dissolve the glucose standard in ultrapure water to prepare a 1 mg / mL standard stock solution, store at 4°C, dilute the standard stock solution into a series of standard working solutions with concentrations of 0.5, 5, 10, 50, 100, and 400 μg / mL, and prepare them before use;

[0129] The standard series working solutions are subjected to derivatization reaction according to step (3);

[0130] The standard series working solutions after the derivatization reaction and the test solution A, test solution B, test solution C, test solution A control, test solution B control and test solution C control were measured by high performance liquid chromatography, and the high performance liquid chromatography conditions were:

[0131] Column: Acclaim TM C30, 5 μm 4.6 × 250 mm HPLC column;

[0132] Mobile phase: a mixed solution of 0.1 mol / L ammonium acetate aqueous solution with a pH of 5.5 and acetonitrile in a volume ratio of 75:25;

[0133] Flow rate: 1.0 mL / min;

[0134] Column temperature: 35°C;

[0135] Detector: UV detector;

[0136] Detection wavelength: 254nm;

[0137] Injection volume: 10 μL;

[0138] The target glucose peak of the standard series working solution was analyzed to obtain the chromatographic peak area. The standard curve was drawn with the concentration of the standard series working solution as the horizontal axis and the chromatographic peak area of ​​the standard series working solution as the vertical axis to obtain the linear regression equation. The target glucose peaks of the test solution A, the test solution B, the test solution C, the test solution A control, the test solution B control and the test solution C control were analyzed to obtain the chromatographic peak areas. The concentrations corresponding to the chromatographic peak areas of the test solution A, the test solution B, the test solution C, the test solution A control, the test solution B control and the test solution C control were calculated by the linear regression equation. The content of yeast β-glucan in the yeast culture sample to be tested was calculated according to formulas (1)-(11). The calculation formula is:

[0139] X D =X A -X B -X C (1)

[0140]

[0141] X 酵母对照-检测值 =X A对照 -X B对照 (7)

[0142] X 谷物对照-检测值 =X c对照 (8)

[0143]

[0144] Where:

[0145] X D -The content of yeast β-glucan in the yeast culture sample to be tested, expressed as mass percentage, %;

[0146] X A -The content of total glucan in the test solution A, expressed as mass percentage, %;

[0147] X B- The content of α-glucan in the test solution B is expressed as mass percentage, %;

[0148] X C- The content of cereal β-glucan in the test solution C is expressed as mass percentage, %;

[0149] X A对照 -The total glucan content in the test solution A control, expressed as mass percentage, %;

[0150] X B对照-The content of α-glucan in the test solution B control, expressed as mass percentage, %;

[0151] X 酵母对照-检测值 -The total glucan content in the test solution A control minus the α-glucan content in the test solution B control, that is, the yeast β-glucan content detected in the test solution A control and the test solution B control, expressed as mass percentage, %;

[0152] X 谷物对照-检测值 - The content of cereal β-glucan in the test solution C control, that is, the content of cereal β-glucan detected in the test solution C control, expressed as mass percentage, %;

[0153] X C对照 -X 谷物对照-检测值 , the content of cereal β-glucan in the test solution C control, that is, expressed as mass percentage, %;

[0154] c A -Concentration corresponding to the chromatographic peak area of ​​glucose in test solution A, μg / mL;

[0155] c B -The concentration corresponding to the chromatographic peak area of ​​glucose in the test solution B, μg / mL;

[0156] c C -Concentration corresponding to the chromatographic peak area of ​​glucose in the test solution C, μg / mL;

[0157] c A对照 -Concentration of glucose chromatographic peak area corresponding to the test solution A control, μg / mL;

[0158] c B对照 -Concentration corresponding to the chromatographic peak area of ​​glucose in the test solution B control, μg / mL;

[0159] c C对照 -Concentration corresponding to the chromatographic peak area of ​​glucose in the test solution C control, μg / mL;

[0160] m A - Total glucan hydrolysis Weigh the mass of the sample to be tested, g;

[0161] m B -α-glucan hydrolysis Weigh the mass of the sample to be tested, g;

[0162] m C - Cereal β-glucan hydrolysis Weigh the mass of the sample to be tested, g;

[0163] m A对照 -Total glucan hydrolysis Weigh the mass of yeast β-glucan reference substance, g;

[0164] m B对照 -α-glucan hydrolysis Weigh the mass of yeast β-glucan reference substance, g;

[0165] m C对照 - Cereal β-glucan hydrolysis Weigh the mass of cereal β-glucan reference substance, g;

[0166] V A -Total glucan hydrolysis sample volume, mL;

[0167] V B -The fixed volume of the α-glucan hydrolyzed sample, mL;

[0168] V C - Volume of the sample to be tested for the hydrolysis of cereal β-glucan, mL;

[0169] V A对照 -Total glucan hydrolyzed yeast β-glucan reference volume, mL;

[0170] V B对照 -α-glucan hydrolyzed yeast β-glucan reference standard volume, mL;

[0171] V C对照 - Cereal β-glucan hydrolyzed cereal β-glucan reference standard volume, mL;

[0172] F 酵母 - Correction factor for low results caused by the destruction or loss of total glucan and α-glucan in the test sample during the test;

[0173] F 谷物 - Correction factor for low results caused by destruction or loss of cereal β-glucan in the test sample during the test;

[0174] P 酵母 -Purity of yeast β-glucan reference substance, %;

[0175] P 谷物 -Purity of cereal β-glucan reference substance, %;

[0176] W 酵母 -Moisture content of yeast β-glucan reference, %;

[0177] W 谷物 - moisture content of the cereal β-glucan reference, %;

[0178] 8.5-The volume dilution factor of the supernatant collected from the hydrolysis of total glucan or cereal β-glucan to prepare the sample solution or reference solution, and then add PMP-methanol solution, sodium hydroxide aqueous solution and hydrochloric acid;

[0179] 4.25-α-glucan hydrolysis, collect the supernatant to prepare the sample solution or reference solution, and then add PMP-methanol solution, sodium hydroxide aqueous solution and hydrochloric acid to dilute the supernatant by volume;

[0180] 0.9 - coefficient for converting glucose content into glucan content;

[0181] X A对照 , X B对照 , X C对照 and X D Perform multiple repeated measurements, and express the measurement results as the arithmetic mean ± standard deviation of the multiple repeated measurement results, retaining 3 significant figures.

[0182] The specific data of the test are:

[0183] Table 1

[0184]

[0185] Table 2

[0186]

[0187] Table 3

[0188]

[0189] Effect test

[0190] 1. Data Statistical Analysis

[0191] GraphPad Prism 9.5.1 was used to analyze and plot the experimental data.

[0192] 2.1 Selection of the amount and ratio of B1 and B2 enzymes

[0193] On the basis of keeping other experimental conditions unchanged, this experiment set up four experimental groups: 0.2mL amyloglucosidase and sucrase group (B1), 0.2mL amyloglucosidase and sucrase + 0.05mL trehalase group (B1+B2), 0.4mL amyloglucosidase and sucrase group (2*B1), 0.4mL amyloglucosidase and sucrase + 0.1mL trehalase group (2*B1+2*B2), and measured the α-glucan concentration in each group of samples. The results are as follows ( Figure 1), the α-glucan concentration in the 2*B1+2*B2 group was 41.653±0.360μg / mL, RSD=0.009%, which was significantly higher than the concentrations in the other three groups (P<0.0001). Compared with the B1 group, the glucan concentrations in the other three groups were significantly increased (P<0.0001). These results show that in addition to substances such as starch and sucrose that are known to contain α-glucan, there are other substances containing α-glucan, including trehalose, in YC. Therefore, in order to achieve thorough hydrolysis, it is necessary to use B1 enzyme and B2 enzyme together for treatment, and the specific addition amount is 0.4mL of B1 enzyme and 0.1mL of B2 enzyme.

[0194] Figure 1 Effects of the addition amount and ratio of B1 and B2 enzymes on the glucose concentration in the sample; B1: 0.2 mL amyloglucosidase and sucrase; 2*B1: 0.4 mL amyloglucosidase and sucrase; B1+B2: 0.2 mL amyloglucosidase and sucrase + 0.05 mL trehalase; 2*B1+2*B2: 0.4 mL amyloglucosidase and sucrase + 0.1 mL trehalase;

[0195] 2.2 Selection of derivation time and extraction times

[0196] Under the premise of keeping other conditions unchanged, this experiment tested the effect of different derivatization reaction times (10min, 30min, 60min, 90min) on the concentration of glucose derivatives. The specific data of the experiment are as follows: at 10min, the concentration was 283.712±12.479μg / mL, and the RSD was 0.044%; at 30min, the concentration rose to 446.461±16.534μg / mL, and the RSD was 0.037%; at 60min, the concentration was 481.578±20.770μg / mL, and the RSD was 0.043%; at 90min, the concentration was 480.124±19.235μg / mL, and the RSD was 0.040%. The results show that ( Figure 2 ), the concentration of glucose derivatives gradually increased with the extension of derivatization reaction time. Compared with the reaction time of 10 min, the concentration of glucose derivatives at 30 min, 60 min, and 90 min increased significantly (P < 0.0001); when the reaction time exceeded 60 min, the concentration of glucose derivatives tended to be stable, so this experiment determined 60 min as the optimal time point for derivatization reaction. This finding shows that the optimization of derivatization reaction time plays an important role in increasing the concentration of derivatives, which may affect the sensitivity and accuracy of the analysis results.

[0197] This experiment selected chloroform as the extraction solvent, using 3 mL each time, and tested the effect of different extraction times (2 times, 3 times, 4 times, 5 times) on the PMP peak area. The results showed that ( Figure 3 ), the PMP peak area gradually decreases with the increase of extraction times. When the extraction times exceed 4 times, the PMP peak area is close to zero, so 4 times is selected as the optimal extraction times. This result shows that the excessive PMP can be effectively removed by the appropriate extraction times, thereby reducing the interference with HPLC analysis and ensuring the accuracy of the analysis results.

[0198] 2.3 Optimization of chromatographic conditions

[0199] This experiment conducted a comparative test of chromatographic columns (i.e. C30 and C18). The results showed that Figure 4 In Figure a, the peak of the glucose PMP derivative separated by the C18 column shows obvious tailing phenomenon, and the peak is wide, which is easily interfered by other peaks. In addition, the peak elution time of the C18 column is 3.640min, which is difficult to distinguish from the peaks of other substances. Figure 4 In Figure b, the C30 column showed a good peak shape with a peak elution time of 7.181 min, which can be effectively distinguished from other similar peaks. Therefore, the C30 column was finally selected for separation.

[0200] 2.4 Linear Relationship

[0201] Under the optimized experimental conditions, the chromatograms of glucose derivatives of glucose standards and experimental group samples are shown in Figure 5 By drawing a glucose standard curve ( Figure 5 Figure b), we observed that in the concentration range of 1 to 400 μg / mL, there was a good linear relationship between glucose concentration and chromatographic peak area, and the fitting equation was y = 77.878x-5.7769, with a correlation coefficient R 2 =1.000. According to the method detection limit (LOD) corresponding to the signal-to-noise ratio (S / N) equal to 3, the detection limit was calculated to be 0.084 mg / L. Similarly, according to the method quantification limit (LOQ) corresponding to the signal-to-noise ratio (S / N) equal to 10, the quantification limit was calculated to be 0.280 mg / L. These results show that this method has high precision and accuracy and can be used as a standard method for quantitative analysis.

[0202] 2.5 Method precision test

[0203] Three different concentrations (10, 50, 100 μg / mL) of glucose standard working solutions were selected and measured under 1.5 chromatographic conditions. Each standard working solution was measured 6 times, and the average value and relative standard deviation of the peak area were calculated.

[0204] The results of the method precision test are listed in Table 4. The RSDs were all less than 1%, indicating that the instrument maintained excellent analytical performance under the current chromatographic conditions.

[0205] Table 4 Method precision test results

[0206]

[0207] 2.6 Spike recovery test

[0208] The fermentation raw materials of this research group were selected as the test group samples, and three different weights of glucose standards were added for recovery test. Six replicates were performed for each spiked level. The operation was performed according to the process of the method in Example 1. The results were measured on the machine under the chromatographic conditions of Example 1, and the average value and relative standard deviation of the peak area were calculated.

[0209] The test results were summarized and the spike recovery was calculated. The results are shown in Table 5. The average recovery of the samples spiked in the range of 1 to 45 mg was 86.670% to 101.863%, and the RSD was less than 1%. This shows that the method has high applicability and feasibility and meets the requirements of routine testing.

[0210] Table 5 Spiked recovery test results

[0211]

[0212] 2.7 Application of the method

[0213] Under the optimized conditions, different types of YC samples produced by 7 different manufacturers were selected as the market sample group for testing. Each sample was repeated 3 times and the results were expressed as the arithmetic mean ± standard deviation.

[0214] The data in Table 6 show that α-glucan and cereal β-glucan are present in all eight yeast cultures, with the α-glucan content ranging from 4.132±0.088% to 33.351±0.175%, and the cereal β-glucan content ranging from

[0215] 0.386±0.007%~1.220±0.059%.

[0216] The yeast β-glucan content in the market sample group ranged from 0.497±0.041% to 1.15±0.119%, and the RSD was less than 1%.

[0217] The experimental group was YC prepared by our research group. The optimized method measured the yeast β-glucan content of 0.814±0.193%, the total glucan content of 21.079±0.995%, the α-glucan content of 19.610±0.819%, and the cereal β-glucan content of 0.655±0.007%, with an RSD of 0.237%. The results showed that there was a high proportion of α-glucan in YC, which may be related to the use of agricultural and sideline products such as corn and soybean meal in the fermentation raw materials.

[0218] Table 6 Detection results of yeast β-glucan in different yeast cultures

[0219]

[0220] Conclusion: The present invention uses enzyme A1, enzyme B1 and enzyme B2, enzyme C1 and enzyme C2 to enzymolyze the glucan in the sample, and establishes an enzymolysis-HPLC detection method for indirectly determining the yeast β-glucan content in YC. This method has high precision, good repeatability and feasibility, and has reference value for the accurate detection of yeast β-glucan content in yeast culture.

[0221] Example 2

[0222] The method for determining the content of yeast β-glucan in yeast culture comprises the following steps:

[0223] (1) The yeast culture to be tested is sampled by the five-point method, and the yeast culture sample to be tested, the yeast β-glucan reference substance of known purity, and the grain β-glucan reference substance of known purity are crushed and passed through a 60-mesh sieve, and stored at -20°C for later use.

[0224] (2) Total glucan hydrolysis: Weigh about 0.1000 g of the pretreated sample and about 0.1000 g of yeast β-glucan reference substance of known purity, add 2.0 mL of 12 mol / L sulfuric acid aqueous solution, ice-bath for 2 h, then add 5 mL of ultrapure water to mix, vortex to mix, the vortex power is 50 W, the speed is 1800 r / min, the oscillation mode is touched, the vortex time is 1 min, then add 5 mL of ultrapure water and boil in water for 2 h, then transfer the solution after boiling in water to 100 mL with sodium acetate buffer of pH 4.5 and concentration of 200 mmol / L. The mixture was centrifuged at 13000 r / min for 5 min and 0.2 mL of the supernatant was collected into a 15 mL centrifuge tube. 0.2 mL of a suspension of exo-1,3-β-glucanase and β-glucosidase was added to the centrifuge tube and heated in a water bath at 40°C for 60 min to obtain 400 μL of sample solution A and 400 μL of reference solution A, respectively.

[0225] α-Glucan hydrolysis: weigh about 0.0900g of the pretreated sample and about 0.0900g of yeast β-glucan reference substance of known purity, add a magnetic stirrer with a diameter of 5mm and a length of 15mm, add 2mL of 1.7mol / L sodium hydroxide aqueous solution and ice water bath, and magnetically stir for 20min at the same time, with a stirring speed of 800r / min. After stirring, add 8mL The pH value is 3.8, and the concentration is 1.2 mol / L sodium acetate buffer for mixing. Then, 0.4 mL of amyloglucosidase and sucrase suspension and 0.1 mL of trehalase suspension are added and heated in a 40°C water bath for 60 min. Finally, the solution heated in a water bath is transferred to a 100 mL volumetric flask with ultrapure water and the volume is made up to 100 mL with ultrapure water. 2 mL of the hydrolyzed solution is centrifuged at a speed of 13000 r / min for 5 min. 0.4 mL of the supernatant is collected in a 15 mL centrifuge tube to obtain 400 μL of sample solution B and 400 μL of reference solution B, respectively.

[0226] Hydrolysis of cereal β-glucan: weigh about 0.1000g of the pretreated sample to be tested and about 0.1000g of a cereal β-glucan reference substance of known purity, add 0.2mL of 50% ethanol aqueous solution and 4mL of pH 6.5, 20mmol / L sodium phosphate buffer, respectively, and boil in a water bath for 5min, then heat in a 50℃ water bath for 5min, then add 0.2mL of endo-1,3-1,4-β-glucan hydrolase suspension, continue to heat in a 50℃ water bath for 60min, and finally transfer the solution heated in a water bath to a 15mL centrifuge tube with pH 4.0, 200mmol / L sodium acetate buffer, and precipitate in a 15mL centrifuge tube with pH 4.0, 200mmol / L sodium acetate buffer. The sample solution to be tested was diluted to 13 mL, and the cereal β-glucan reference solution was diluted to 15 mL. After the volume was diluted, 2 mL of the hydrolyzed solution was centrifuged at a speed of 13000 r / min for 5 min. 0.2 mL of the supernatant was collected and transferred to a 15 mL centrifuge tube. 0.2 mL of β-glucosidase suspension was added to the centrifuge tube, and the tube was heated in a 50°C water bath for 10 min to obtain 400 μL of sample solution C and 400 μL of reference solution C, respectively.

[0227] (3) Take the sample solution A, sample solution B, sample solution C, reference solution A, reference solution B and reference solution C obtained in step (2), add 400 μL of PMP-methanol solution with a PMP concentration of 0.5 mol / L and 400 μL of sodium hydroxide aqueous solution with a concentration of 0.3 mol / L, respectively, and heat in a water bath at 70°C for 60 min, then add 500 μL of 0.3 mol / L hydrochloric acid, and then repeat the extraction with 3 mL of chloroform four times each time, finally take the upper aqueous phase and centrifuge it at a centrifugal speed of 10000 r / min for 2 min, collect the supernatant and filter it with a 0.22 μm syringe filter to obtain test solution A, test solution B, test solution C, test solution A control, test solution B control and test solution C control.

[0228] (4) Drawing and determination of standard curve:

[0229] Dissolve the glucose standard in ultrapure water to prepare a 1 mg / mL standard stock solution, store at 4°C, dilute the standard stock solution into a series of standard working solutions with concentrations of 0.5, 5, 10, 50, 100, and 400 μg / mL, and prepare them before use;

[0230] The standard series working solutions are subjected to derivatization reaction according to step (3);

[0231] The standard series working solutions after the derivatization reaction and the test solution A, test solution B, test solution C, test solution A control, test solution B control and test solution C control were measured by high performance liquid chromatography, and the high performance liquid chromatography conditions were:

[0232] Column: Acclaim TM C30, 5 μm 4.6 × 250 mm HPLC column;

[0233] Mobile phase: a mixed solution of 0.1 mol / L ammonium acetate aqueous solution with a pH of 5.5 and acetonitrile in a volume ratio of 75:25;

[0234] Flow rate: 1.0 mL / min;

[0235] Column temperature: 35°C;

[0236] Detector: UV detector;

[0237] Detection wavelength: 254nm;

[0238] Injection volume: 10 μL;

[0239] The target glucose peak of the standard series working solution was analyzed to obtain the chromatographic peak area. The standard curve was drawn with the concentration of the standard series working solution as the horizontal axis and the chromatographic peak area of ​​the standard series working solution as the vertical axis to obtain the linear regression equation. The target glucose peaks of the test solution A, the test solution B, the test solution C, the test solution A control, the test solution B control and the test solution C control were analyzed to obtain the chromatographic peak areas. The concentrations corresponding to the chromatographic peak areas of the test solution A, the test solution B, the test solution C, the test solution A control, the test solution B control and the test solution C control were calculated by the linear regression equation. The yeast β-glucan content in the yeast culture sample to be tested was calculated according to formulas (1)-(11).

[0240] X A对照 , X B对照 , X C对照 and X D Perform multiple repeated measurements, and express the measurement results as the arithmetic mean ± standard deviation of the multiple repeated measurement results, retaining 3 significant figures.

[0241] The specific data of the test are:

[0242] Table 7

[0243]

[0244] Table 8

[0245]

[0246] Table 9

[0247]

[0248] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for determining the content of yeast β-glucan in yeast culture, characterized in that: The following steps are involved: (1) Pretreatment: The yeast culture sample to be tested, the yeast β-glucan reference substance of known purity, and the grain β-glucan reference substance of known purity are crushed and sieved for later use; (2) Total glucan hydrolysis: Weigh the pretreated sample to be tested and a yeast β-glucan reference substance of known purity, add sulfuric acid aqueous solution to each of them, place in an ice water bath, then add ultrapure water to place in a boiling water bath, transfer the solution after the boiling water bath to a volumetric flask with sodium acetate buffer, then add sodium hydroxide aqueous solution to the volumetric flask for neutralization, finally make up to volume with sodium acetate buffer, centrifuge the hydrolyzed solution, collect the supernatant into a centrifuge tube, add a suspension of exo-1,3-β-glucanase and β-glucosidase to the centrifuge tube, and heat in a water bath to obtain sample solution A and reference substance solution A; α-glucan hydrolysis: weigh the pretreated sample to be tested and the yeast β-glucan reference substance of known purity, add sodium hydroxide aqueous solution to each of them, place in an ice water bath, and stir magnetically, then add sodium acetate buffer to mix, then add a suspension of amyloglucosidase and sucrase and a suspension of trehalase to heat in a water bath, finally transfer the solution after water bath heating to a volumetric flask with ultrapure water and make up to volume, centrifuge the hydrolyzed solution, collect the supernatant into a centrifuge tube, and obtain sample solution B and reference substance solution B; Hydrolysis of cereal β-glucan: Weigh the pretreated sample to be tested and the cereal β-glucan reference substance of known purity, add ethanol aqueous solution and sodium phosphate buffer respectively, place in a boiling water bath, then heat in a water bath, add the suspension of endo-1,3-1,4-β-glucan hydrolase, continue to heat in a water bath, finally transfer the solution heated in a water bath to a volumetric flask with sodium acetate buffer and make up to volume, centrifuge the hydrolyzed solution, collect the supernatant into a centrifuge tube, add the suspension of β-glucosidase into the centrifuge tube, and heat in a water bath to obtain sample solution C and reference substance solution C; (3) Derivatization reaction: take the sample solution A, sample solution B, sample solution C, reference solution A, reference solution B, and reference solution C obtained in step (2), add PMP-methanol solution and sodium hydroxide aqueous solution respectively, heat in a water bath, then add hydrochloric acid, and extract repeatedly with chloroform for multiple times, finally take the upper aqueous phase for centrifugation, collect the supernatant and filter, and obtain test solution A, test solution B, test solution C, test solution A control, test solution B control, and test solution C control; (4) Drawing and determination of standard curve: Dissolve the glucose standard in ultrapure water to prepare a 1 mg / mL standard stock solution, store at 4°C, dilute the standard stock solution into a series of standard working solutions with concentrations of 0.5, 5, 10, 50, 100, and 400 μg / mL, and prepare them before use; The standard series working solutions are subjected to derivatization reaction according to step (3); The standard series working solutions after the derivatization reaction and the test solution A, test solution B, test solution C, test solution A control, test solution B control and test solution C control were measured by high performance liquid chromatography, and the high performance liquid chromatography conditions were: Column: Acclaim TM C30, 5 μm 4.6 × 250mm HPLC column; Mobile phase: a mixed solution of 0.1 mol / L ammonium acetate aqueous solution with a pH of 5.5 and acetonitrile in a volume ratio of 75:25; Flow rate: 1.0 mL / min; Column temperature: 35°C; Detector: UV detector; Detection wavelength: 254nm; Injection volume: 10 μL; The target glucose peak of the standard series working solution was analyzed to obtain the chromatographic peak area. The standard curve was drawn with the concentration of the standard series working solution as the horizontal axis and the chromatographic peak area of ​​the standard series working solution as the vertical axis to obtain the linear regression equation. The target glucose peaks of the test solution A, the test solution B, the test solution C, the test solution A control, the test solution B control and the test solution C control were analyzed to obtain the chromatographic peak areas. The concentrations corresponding to the chromatographic peak areas of the test solution A, the test solution B, the test solution C, the test solution A control, the test solution B control and the test solution C control were calculated by the linear regression equation. The content of yeast β-glucan in the yeast culture sample to be tested was calculated according to formulas (1)-(11). The calculation formula is: X D =X A -X B -X C (1) X 酵母对照-检测值 =X A对照 -X B对照 (7) X 谷物对照-检测值 =X C对照 (8) Where: X D -The content of yeast β-glucan in the yeast culture sample to be tested, expressed as mass percentage, %; X A -The content of total glucan in the test solution A, expressed as mass percentage, %; X B- The content of α-glucan in the test solution B is expressed as mass percentage, %; X C- The content of cereal β-glucan in the test solution C is expressed as mass percentage, %; X A对照 -The total glucan content in the test solution A control, expressed as mass percentage, %; X B对照 -The content of α-glucan in the test solution B control, expressed as mass percentage, %; X 酵母对照-检测值 -The total glucan content in the test solution A control minus the α-glucan content in the test solution B control, that is, the yeast β-glucan content detected in the test solution A control and the test solution B control, expressed as mass percentage, %; X 谷物对照-检测值 - The content of cereal β-glucan in the test solution C control, that is, the content of cereal β-glucan detected in the test solution C control, expressed as mass percentage, %; X C对照 -X 谷物对照-检测值 , the content of cereal β-glucan in the test solution C control, that is, expressed as mass percentage, %; c A -Concentration corresponding to the chromatographic peak area of ​​glucose in test solution A, μg / mL; c B -The concentration corresponding to the chromatographic peak area of ​​glucose in the test solution B, μg / mL; c C -Concentration corresponding to the chromatographic peak area of ​​glucose in the test solution C, μg / mL; c A对照 -Concentration of glucose chromatographic peak area corresponding to the test solution A control, μg / mL; c B对照 -Concentration corresponding to the chromatographic peak area of ​​glucose in the test solution B control, μg / mL; c C对照 -Concentration corresponding to the chromatographic peak area of ​​glucose in the test solution C control, μg / mL; m A - Total glucan hydrolysis Weigh the mass of the sample to be tested, g; m B -α-glucan hydrolysis Weigh the mass of the sample to be tested, g; m C - Cereal β-glucan hydrolysis Weigh the mass of the sample to be tested, g; m A对照 -Total glucan hydrolysis Weigh the mass of yeast β-glucan reference substance, g; m B对照 -α-glucan hydrolysis Weigh the mass of yeast β-glucan reference substance, g; m C对照 - Cereal β-glucan hydrolysis Weigh the mass of cereal β-glucan reference substance, g; V A -Total glucan hydrolysis sample volume, mL; V B -The fixed volume of the α-glucan hydrolyzed sample, mL; V C - Volume of the sample to be tested for the hydrolysis of cereal β-glucan, mL; V A对照 -Total glucan hydrolyzed yeast β-glucan reference volume, mL; V B对照 -α-glucan hydrolyzed yeast β-glucan reference standard volume, mL; V C对照 - Cereal β-glucan hydrolyzed cereal β-glucan reference standard volume, mL; F 酵母 - Correction factor for low results caused by the destruction or loss of total glucan and α-glucan in the test sample during the test; F 谷物 - Correction factor for low results caused by destruction or loss of cereal β-glucan in the test sample during the test; P 酵母 -Purity of yeast β-glucan reference substance, %; P 谷物 -Purity of cereal β-glucan reference substance, %; W 酵母 -Moisture content of yeast β-glucan reference, %; W 谷物 - moisture content of the cereal β-glucan reference, %; 8.5-The volume dilution factor of the supernatant collected from the hydrolysis of total glucan or cereal β-glucan to prepare the sample solution or reference solution, and then add PMP-methanol solution, sodium hydroxide aqueous solution and hydrochloric acid; 4.25-α-glucan hydrolysis, collect the supernatant to prepare the sample solution or reference solution, and then add PMP-methanol solution, sodium hydroxide aqueous solution and hydrochloric acid to dilute the supernatant by volume; 0.9 - coefficient for converting glucose content into glucan content; X A对照 , X B对照 , X C对照 and X D Perform multiple repeated measurements, and express the measurement results as the arithmetic mean ± standard deviation of the multiple repeated measurement results, retaining 3 significant figures.

2. A method for determining the content of yeast β-glucan in yeast culture according to claim 1, characterized in that: In step (1), the yeast culture to be tested is sampled by the five-point method, and the yeast culture sample to be tested, the yeast β-glucan reference substance of known purity, and the grain β-glucan reference substance of known purity are crushed, passed through a 60-mesh sieve, and stored at -20°C for later use.

3. A method for determining the content of yeast β-glucan in yeast culture according to claim 1, characterized in that: In step (2), total glucan hydrolysis: weigh 0.0500-0.1200g of the pretreated sample to be tested and 0.0500-0.1200g of yeast β-glucan reference substance of known purity, add 2.0mL of 12mol / L sulfuric acid aqueous solution to each of them, and then place them in an ice water bath for 2h, then add 10mL of ultrapure water and place them in a boiling water bath for 2h, then transfer the solution after the boiling water bath to a 100mL volumetric flask with a pH 4.5, 200mmol / L sodium acetate buffer, and then add 6mL of 8mol / L The mixture was neutralized with sodium hydroxide aqueous solution, and finally the volume was made up to 100 mL with a sodium acetate buffer having a pH value of 4.5 and a concentration of 200 mmol / L. 1-2 mL of the hydrolyzed solution was centrifuged at a speed of 13000 r / min for 5 min. 0.2 mL of the supernatant was collected into a 15 mL centrifuge tube. 0.2 mL of a suspension of exo-1,3-β-glucanase and β-glucosidase was added to the centrifuge tube, and then heated in a water bath at 40°C for 60 min to obtain 400 μL of sample solution A and 400 μL of reference solution A, respectively.

4. A method for determining the content of yeast β-glucan in yeast culture according to claim 1, characterized in that: In step (2), α-glucan hydrolysis: weigh 0.0500-0.1200g of the pretreated sample to be tested and 0.0500-0.1200g of yeast β-glucan reference substance of known purity, add a magnetic stirring bar with a diameter of 5mm and a length of 15mm, add 2mL of 1.7mol / L sodium hydroxide aqueous solution and ice water bath, and magnetically stir for 20min while ice water bath, the stirring speed is 600-1000r / min, and after stirring, add 8mL The pH value is 3.8, and the concentration is 1.2 mol / L sodium acetate buffer for mixing. Then, 0.4 mL of amyloglucosidase and sucrase suspension and 0.1 mL of trehalase suspension are added and heated in a 40°C water bath for 60 min. Finally, the solution heated in a water bath is transferred to a 100 mL volumetric flask with ultrapure water and the volume is fixed to 100 mL. 2-4 mL of the hydrolyzed solution is centrifuged at a speed of 13000 r / min for 5 min. 0.4 mL of the supernatant is collected in a 15 mL centrifuge tube to obtain 400 μL of sample solution B and 400 μL of reference solution B, respectively.

5. The method for determining the content of yeast β-glucan in yeast culture according to claim 1, characterized in that: In step (2), hydrolysis of cereal β-glucan: weigh 0.0500-0.1200 g of the pretreated sample to be tested and 0.0500-0.1200 g of a cereal β-glucan reference substance of known purity, add 0.2 mL of 50% ethanol aqueous solution and 4 mL of The mixture was boiled in a water bath for 5 min with a pH value of 6.5 and a concentration of 20 mmol / L sodium phosphate buffer, then heated in a water bath at 50°C for 5 min, and then 0.2 mL of a suspension of endo-1,3-1,4-β-glucan hydrolase was added, and the mixture was heated in a water bath at 50°C for 60 min. Finally, the solution heated in a water bath was transferred to a 10-50 mL centrifuge tube with a pH value of 4.0 and a concentration of 200 mmol / L sodium acetate buffer and fixed to 10-50 mL. 2-4 mL of the hydrolyzed solution was centrifuged at a speed of 13000 r / min for 5 min. 0.2 mL of the supernatant was collected and transferred to a 15 mL centrifuge tube. 0.2 mL of a suspension of β-glucosidase was added to the centrifuge tube and heated in a water bath at 50°C for 10 min to obtain 400 μL of a sample solution C and 400 μL of a reference solution C, respectively.

6. A method for determining the content of yeast β-glucan in yeast culture according to claim 1, characterized in that: In step (3), 400 μL of each of the sample solution A, sample solution B, sample solution C, reference solution A, reference solution B, and reference solution C obtained in step (2) are taken, 400 μL of PMP-methanol solution with a PMP concentration of 0.5 mol / L and 400 μL of sodium hydroxide aqueous solution with a concentration of 0.3 mol / L are added, and then heated in a water bath at 70°C for 60 min, and then 500 μL of hydrochloric acid with a concentration of 0.3 mol / L is added, and then the extraction is repeated 4 times with 3 mL of chloroform each time, and finally the upper aqueous phase is taken for centrifugation at a centrifugal speed of 10000 r / min and a centrifugal time of 2 min. The supernatant is collected and filtered through a 0.22 μm syringe filter to obtain test solution A, test solution B, test solution C, test solution A control, test solution B control, and test solution C control.

7. A method for determining the content of yeast β-glucan in yeast culture according to claim 1, characterized in that: The preparation method of the suspension of exo-1,3-β-glucanase and β-glucosidase comprises the following steps: To 2.0 mL of a suspension having an enzyme concentration of 100 U / mL of exo-1,3-β-glucanase and an enzyme concentration of 20 U / mL of β-glucosidase, 9 mL of a sodium acetate buffer having a pH of 4.5 and a concentration of 200 mmol / L was added to dilute the suspension to 11 mL, and the suspension was divided into equal portions for storage; The suspension of amyloglucosidase and sucrase is: A suspension of amyloglucosidase and sucrase in a 50% glycerol aqueous solution as solvent (20 mL in total), wherein the enzyme activity of amyloglucosidase is 1630 U / mL and the enzyme activity of sucrase is 500 U / mL, is used directly; Trehalase suspension is: Trehalase suspension, 5 mL, used directly; The preparation method of the suspension of endo-1,3-1,4-β-glucanohydrolase comprises the following steps: Dilute 1.0 mL of the initial suspension of endo-1,3-1,4-β-glucanohydrolase to 20.0 mL with a sodium phosphate buffer having a pH of 6.5 and a concentration of 20 mmol / L, so that the final enzyme concentration of the endo-1,3-1,4-β-glucanohydrolase is 50 U / mL, and store the suspension in equal portions; The preparation method of the suspension of β-glucosidase comprises the following steps: 1.0 mL of the initial suspension of β-glucosidase was diluted to 20.0 mL with a pH 4.0, 50 mmol / L sodium acetate buffer to make the final enzyme concentration of β-glucosidase 2 U / mL, and the suspension was stored in equal portions.

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