A method for determining the content of alpha-mannan in yeast culture

Abstract

The present invention discloses a method for determining the content of α-mannan in yeast culture, which belongs to the field of chemical analysis technology. The method comprises: pretreatment; hydrolysis; derivatization reaction; drawing and determining a standard curve. The present invention is used to quantitatively analyze the content of α-mannan in YC. The method will provide an important reference basis for the further development and preparation of YC, and contribute more valuable scientific data to the development of related fields. With the help of this method, we can more accurately evaluate the content of α-mannan in YC, thereby ensuring the quality of the product and the accuracy of the functional declaration.

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 alpha-mannan in yeast culture. Background Art

[0002] Yeast Culture (YC) is a microecological preparation produced by a special fermentation process. Its main components include cell metabolites, denatured culture medium and a small amount of inactivated yeast cells. Studies have shown that α-mannan is an antigenic active substance derived from yeast cell walls. It belongs to the polysaccharide class and has the characteristics of acid resistance, high temperature resistance and pressure resistance. It is one of the main effective ingredients in YC. In animal nutrition, α-mannan has many important biological functions, including improving the body's immunity, promoting animal growth, regulating the balance of intestinal flora, enhancing the body's antioxidant capacity and adsorbing mycotoxins. Due to these functions, α-mannan has been widely used in poultry, livestock and aquatic industries.

[0003] Mannans present in plants are β-mannans, which are important members of the hemicellulose family. Their core structure is always composed of a main chain of only mannose residues connected by β-1,4-glycosidic bonds, which may be accompanied by a combination of mannose and mannose residues and α-1,6-linked galactose residue side chains. β-Mannans can be subdivided into four subclasses: linear mannans, glucomannans, galactomannans and galactoglucomannans. These mannans play a key role in the growth, maturation and senescence of plants by participating in the metabolism of the cell wall. They not only serve as food reserves, but also play an important role in the retention of water in cells. β-Mannans are usually enriched in the endosperm of leguminous plants and are commonly found in various cereals that form the basis of livestock feed. They may interfere with the digestion and utilization of nutrients and are therefore considered an anti-nutrient. However, the mannan present in yeast is α-mannan, i.e. yeast mannan, which is a highly branched polymer with an α-1,6-linked main chain and α-1,2- and α-1,3-linked mannose branches, which is different from the mannan structure in plants. The degree of branching of the mannan molecule can affect its ability to interact with immune cells, and may show stronger interactions with specific receptors on immune cells, resulting in stronger or more targeted immune responses. Similarly, the molecular weight of mannan can affect its function as a prebiotic. Due to its special biological activity, α-mannan is used as a prebiotic in animal husbandry and nutritional supplements and is an important nutrient. Given the complex matrix characteristics of YC, in which α-mannan and β-mannan are present at the same time, the accurate determination of α-mannan content in YC has always been a research difficulty.

[0004] Studies have shown that α-mannan concentration is a key indicator for evaluating the quality of yeast cultures. With the widespread application of YC in the livestock industry, it is particularly important to accurately determine the content of its active ingredient α-mannan. However, there is currently a lack of standard detection methods for accurately determining the content of α-mannan in yeast cultures at home and abroad. Therefore, in order to evaluate the quality of YC, it is important to quantitatively analyze the content of α-mannan, the main active ingredient in YC, to safeguard the interests of users, improve product quality, and ensure the safety of livestock products. It is of great significance to develop a method for accurately determining the content of α-mannan in yeast cultures.

[0005] The reported methods for detecting mannan mainly include colorimetry, enzymatic method and high performance liquid chromatography. The colorimetric method can detect compounds with sugar structures, but its selectivity is poor. The enzymatic method is mainly used to detect and analyze mannan from plant sources, but its analysis process is time-consuming, cumbersome and has poor repeatability. High performance liquid chromatography hydrolyzes mannan into monosaccharide-mannose through acid hydrolysis, then undergoes pre-column or post-column derivatization, separates using a C18 chromatographic column, and detects using an ultraviolet or fluorescence detector. After derivatization, the separation selectivity and detection sensitivity of monosaccharides are significantly enhanced. However, in YC, the acid hydrolysis step of high performance liquid chromatography will hydrolyze α-mannan and β-mannan at the same time, resulting in the determination of the total mannan content, which makes the measured value higher.

[0006] Therefore, how to develop a method for determining the content of α-mannan in yeast culture is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a method for determining the content of α-mannan in yeast culture.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A method for determining the content of α-mannan in a yeast culture comprises the following steps:

[0010] (1) Pretreatment: grind the yeast culture sample to be tested and the yeast mannan reference substance of known purity, then sieve them for later use;

[0011] (2) Hydrolysis: Weigh the pretreated sample and yeast mannan reference substance of known purity, place them in test tubes, add glacial sulfuric acid, shake and mix, and immediately incubate in ice water. Then add water to each test tube, mix and incubate in a boiling water bath. Transfer the solution in the test tube containing the sample to a centrifuge tube using a water-washing bottle containing MES buffer solution. Transfer the solution in the test tube containing the yeast mannan reference substance to a conical flask using a water-washing bottle containing MES buffer solution. Then add sodium hydroxide aqueous solution to the centrifuge tube and conical flask respectively, and make up the volume with MES buffer solution to the mark but not exceeding and close to the mark. Adjust the pH to 4.5-5 with or without sodium hydroxide aqueous solution. 5 (If the pH of the solution in the centrifuge tube or conical flask is 4.5-5.5 after the MES buffer solution is used to make the volume up to the mark but not exceeding and close to the scale, then the pH is no longer adjusted with the sodium hydroxide aqueous solution. If the pH of the solution in the centrifuge tube or conical flask is not 4.5-5.5 after the MES buffer solution is used to make the volume up to the mark but not exceeding and close to the scale, then the pH is adjusted to 4.5-5.5 with the sodium hydroxide aqueous solution). Finally, make the volume up to the mark with the MES buffer solution, centrifuge the hydrolyzed solution, collect the supernatant to the bottom of the centrifuge tube, add the dilution of the α-mannosidase suspension to the centrifuge tube, mix well, and place in a constant temperature water bath to obtain the sample solution and the reference solution respectively.

[0012] (3) Derivatization reaction: PMP-methanol solution and sodium hydroxide aqueous solution were added to the sample solution and reference solution obtained in step (2) respectively, and then heated in a water bath. Hydrochloric acid was then added, and the extraction was repeated several times with chloroform. Finally, the upper aqueous phase was centrifuged and the supernatant was collected by needle filter to obtain the sample solution and the reference solution.

[0013] (4) Drawing and determination of standard curve:

[0014] Dissolve the mannose standard in 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;

[0015] The standard series working solutions were subjected to derivatization reaction according to step (3);

[0016] The standard series working solutions, test solution and test control solution after the derivatization reaction were measured by high performance liquid chromatography, and the high performance liquid chromatography conditions were:

[0017] Column: Acclaim TM C30, 5 μm 4.6 × 250 mm HPLC column;

[0018] 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;

[0019] Flow rate: 1.0 mL / min;

[0020] Column temperature: 35 °C;

[0021] Detector: UV detector;

[0022] Detection wavelength: 254 nm;

[0023] Injection volume: 10 μL;

[0024] The mannose target 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 mannose target peak of the test solution and the test control solution was analyzed to obtain the chromatographic peak area. The concentration corresponding to the mannose chromatographic peak area of ​​the test solution and the test control solution was calculated by the linear regression equation. The content of α-mannan in the yeast culture sample to be tested was calculated according to formula (1)-(3). The calculation formula is:

[0025]

[0026] Where:

[0027] X i -The content of α-mannan in the sample to be tested, expressed as mass percentage, %;

[0028] X i对照 -The content of yeast mannan in the yeast mannan reference substance to be tested, expressed as mass percentage, %;

[0029] c1-Concentration corresponding to the chromatographic peak area of ​​mannose in the test solution, μg / mL;

[0030] c2-Concentration corresponding to the chromatographic peak area of ​​mannose in the test sample control solution, μg / mL;

[0031] m1-the mass of the sample to be tested after hydrolysis, g;

[0032] m2-the mass of the hydrolyzed yeast mannan reference substance, g;

[0033] V1-volume of the sample to be hydrolyzed, mL;

[0034] V2- volume of hydrolyzed yeast mannan reference substance, mL;

[0035] F-Correction factor for low results caused by the destruction or loss of α-mannan in the test sample during the test;

[0036] Purity of P-yeast mannan reference substance, %;

[0037] W- moisture content of yeast mannan reference substance, %;

[0038] n-i.e. 8.5, the volume dilution multiple of the supernatant after collecting the supernatant to prepare the sample solution or the reference solution, adding α-mannosidase, and then adding PMP-methanol solution, sodium hydroxide aqueous solution and hydrochloric acid;

[0039] 0.9 - coefficient for converting mannose content into mannan content;

[0040] X i , X i对照 Repeat the determination with F and X. i The measurement results were expressed as the arithmetic mean ± standard deviation of multiple repeated measurement results, with 3 significant figures retained.

[0041] 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 and a yeast mannan reference substance of known purity are crushed and passed through a 60-mesh sieve for later use.

[0042] Further, in step (2), 0.0600-0.1200 g of the pretreated sample to be tested and 0.0800-0.1100 g of yeast mannan reference substance of known purity are weighed, placed in test tubes, 2.0 mL of 12 mol / L glacial sulfuric acid is added, and the mixture is shaken and mixed, and then immediately placed in ice water for incubation for 30 min. Then 10 mL of water is added to each test tube, and after mixing, the mixture is placed in a boiling water bath for incubation for 30 min. The solution in the test tube containing the sample to be tested is transferred to a 50 mL centrifuge tube using a water washing bottle containing a pH 5.5, 50 mmol / L MES buffer solution. Then 6 mL of an 8 mol / L sodium hydroxide aqueous solution is added to the centrifuge tube, and the volume is adjusted to not more than and close to the 50 mL mark with a pH 5.5, 50 mmol / L MES buffer solution. The pH is adjusted to 4.5-5.5 with or without a sodium hydroxide aqueous solution, and finally the volume is adjusted to 50 mL mark with ultrapure water. The solution in the mannan reference test tube was transferred to a 100mL conical flask with a washing bottle containing a pH 5.5, 50mmol / L MES buffer solution, and then 6mL of an 8mol / L sodium hydroxide aqueous solution was added to the conical flask, and the volume was fixed to not exceeding and close to the 100mL mark with a pH 5.5, 50mmol / L MES buffer solution, and the pH was adjusted to 4.5-5.5 with or without sodium hydroxide aqueous solution, and the pH was adjusted to 5.5. Finally, the volume was fixed to 100mL with ultrapure water, and 1-2mL of the hydrolyzed solution was centrifuged at a speed of 13000r / min and a centrifugal time of 5min. 0.2mL of the supernatant was collected to the bottom of a 15mL centrifuge tube, and 0.2mL of the dilution of the α-mannosidase suspension was added to the centrifuge tube, mixed thoroughly, and placed in a 37°C constant temperature water bath for 17h to obtain 400μL of sample solution and 400μL of reference solution, respectively.

[0043] Further, in step (3), 400 μL of the sample solution and the reference solution obtained in step (2) are taken, 400 μL of a PMP-methanol solution with a PMP concentration of 0.5 mol / L and 400 μL of a 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 10 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 needle filter to obtain a test solution and a test control solution.

[0044] Furthermore, the preparation method of the dilution of the α-mannosidase suspension comprises the following steps:

[0045] Suspend 1 mg of α-mannosidase with an enzyme activity ≥15 U / mg in a mixed aqueous solution of 3 mol / L (NH4)2SO4 and 0.1 mmol / L zinc acetate to obtain 230-250 μL of α-mannosidase suspension with a pH of 7.5. Add 1 mL of ultrapure water to the α-mannosidase suspension for dilution, store at 4°C, and use within one week.

[0046] Furthermore, in step (2), the method for preparing a 50 mmol / L MES buffer solution having a pH of 5.5 and a concentration of 50 mmol / L comprises the following steps:

[0047] Accurately weigh 9.76 g of MES, add it to 900 mL of ultrapure water to dissolve, adjust the pH to 5.5, finally make up to 1 L, and store at 4 °C.

[0048] Beneficial effects of the present invention: The present invention is used to quantitatively analyze the content of α-mannan in YC. This method will provide an important reference for the further development and preparation of YC, and contribute more valuable scientific data to the development of related fields. With the help of this method, we can more accurately evaluate the content of α-mannan in YC, thereby ensuring the quality of the product and the accuracy of the functional declaration. The test results show that mannose has a good linear relationship in the concentration range of 1~400μg / mL, the correlation coefficient is 1.000, the detection limit is 0.063mg / L, the quantitative limit is 0.208mg / L, the average spiked recovery is 87.661%~93.821%, and the RSD is less than 1%. These data show that the method established by the present invention has high precision, good repeatability and feasibility, and is suitable for the detection of α-mannan in yeast culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Effect of enzymatic hydrolysis time on the concentration of mannose derivatives in samples;

[0050] Figure 2 The results of the effect of derivatization reaction time on the concentration of mannose derivatives in samples;

[0051] Figure 3 Results of the effect of extraction times on PMP peak area;

[0052] Figure 4 In the figure, a is the chromatogram of mannose PMP derivatives in mannose standards and test group samples, and b is the standard curve of mannose. DETAILED DESCRIPTION

[0053] 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.

[0054] 1. Instruments and equipment

[0055] Agilent 1260 Infinity Ⅱ HPLC system (Agilent, USA); chromatographic column: Acclaim TM C30 (5μm, 4.6×250mm) (Thermo Fisher Scientific (China) Co., Ltd.); ME204E electronic analytical balance (accuracy 0.0001g, Mettler-Toledo Technologies (China) Co., Ltd.); Milli-Q ultrapure water system (Merck, Germany); high-speed refrigerated centrifuge (Hunan Duoheng Instrument Equipment Co., Ltd.); microcentrifuge (Epend Centrifuge 5425, Shanghai Gaosiwei Medical Technology Co., Ltd.); vortex mixer (Vortex-Genie 2 SI-0246, Hangzhou Ruicheng Instrument Co., Ltd.); high-speed pulverizer (Zhejiang Yili Industry and Trade Co., Ltd.); magnetic stirrer (Thermo Fisher Scientific (China) Co., Ltd.); pH meter (accuracy 0.01, Mettler-Toledo Technologies (China) Co., Ltd.).

[0056] 2. Reagents and raw materials

[0057] Mannose standard, analytical grade acetonitrile, and 1-phenyl-3-methyl-5-pyrazolone (hereinafter referred to as PMP) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; yeast mannan standard (purity ≥ 99.9%, yeast-derived α-mannan) and α-mannosidase (derived from Jack Bean) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; 2-(N-morpholinoline)ethanesulfonic acid (hereinafter referred to as MES) was purchased from Saiguo Biotechnology Co., Ltd.; sulfuric acid, hydrochloric acid, sodium hydroxide, and dichloromethane were purchased from Fuchen (Tianjin) Chemical Reagent Co., Ltd.;

[0058] Yeast mannan reference substance: Use the fermentation base of yeast culture as blank raw material, add yeast mannan standard substance to prepare yeast mannan reference substance with known content.

[0059] The fermentation base material is composed of the following raw materials in percentage by mass:

[0060] Bran 20%, sprayed corn husk 12%, corn 10%, rice bran (second bran) 10%, cottonseed meal 10%, corn germ meal 28%, soybean meal 10%.

[0061] The preparation method of the dilution of the α-mannosidase suspension comprises the following steps:

[0062] Suspend 1 mg of α-mannosidase with an enzyme activity ≥15 U / mg in a mixed aqueous solution of 3 mol / L (NH4)2SO4 and 0.1 mmol / L zinc acetate to obtain 240 μL of α-mannosidase suspension with a pH of 7.5. Add 1 mL of ultrapure water to the α-mannosidase suspension for dilution, store at 4°C, and use within one week. It is hereinafter referred to as enzyme A.

[0063] The preparation method of the LMES buffer solution with a pH of 5.5 and a concentration of 50 mmol / L comprises the following steps:

[0064] Accurately weigh 9.76 g of MES, add it to 900 mL of ultrapure water to dissolve, adjust the pH to 5.5, finally make up to 1 L, and store at 4 °C.

[0065] Example 1

[0066] The method for determining the content of α-mannan in yeast culture comprises the following steps:

[0067] (1) The yeast culture fermentation raw material, i.e. the blank group, was in dry state with a water content of approximately 0%. The weighed masses were: 0.0804 g, 0.0787 g, and 0.0795 g, respectively;

[0068] The mass of yeast mannan standard was weighed: 0.0306 g, with a water content of 0%. After being dissolved in 1.2 mL of ultrapure water, it was divided into three portions, 400 μL each (i.e., 0.0102 g yeast mannan standard each) and added to the above three blank groups to obtain the following three groups of yeast mannan reference substances:

[0069] The actual masses of yeast mannan reference substances are m2: 0.0906 g, 0.0889 g, and 0.0897 g;

[0070] The theoretical contents of yeast mannan reference substances are: 11.258%, 11.474%, 11.371%; namely, P (100%-W) values;

[0071] The yeast culture to be tested is sampled by the five-point method, and the yeast culture sample to be tested and a yeast mannan reference substance of known purity are crushed and passed through a 60-mesh sieve for later use.

[0072] (2) Weigh the pretreated sample and yeast mannan reference substance of known purity, place them in test tubes, add 2.0 mL of 12 mol / L sulfuric acid aqueous solution stored in a 4°C refrigerator, vortex and mix well. Vortex at a power of 50 W and a speed of 1800 r / min. Touch the oscillation mode. Vortex for 1 min. Immediately place in ice water and incubate for 30 min. Repeat vortexing every 10 min. Then add 10 mL of water to each test tube, mix well, and incubate in a boiling water bath for 30 min. The solution in the test tube containing the sample to be tested was transferred to a 50mL centrifuge tube using a water-washing bottle containing a pH 5.5, 50mmol / LMES buffer solution, and then 6mL of an 8mol / L sodium hydroxide aqueous solution was added to the centrifuge tube. Finally, the pH was adjusted to 5.5 with an 8mol / L sodium hydroxide aqueous solution, and finally the pH was adjusted to 5.5 with a pH 5.5, 50mmol / LMES buffer solution. The solution in the test tube containing the yeast mannan reference substance was transferred to a 100mL conical flask using a water-washing bottle containing a pH 5.5, 50mmol / LMES buffer solution, and then the conical flask was added. 6mL of 8mol / L sodium hydroxide aqueous solution, and finally made up to not exceeding and close to the 100mL mark with pH5.5, 50mmol / LMES buffer solution, adjust the pH to 5.5 with 8mol / L sodium hydroxide aqueous solution, and finally made up to 100mL mark with pH5.5, 50mmol / LMES buffer solution, take 2mL of the hydrolyzed solution and centrifuge at a speed of 13000r / min and a centrifugal time of 5min, collect 0.2mL of the supernatant to the bottom of a 15mL centrifuge tube, add 0.2mL of the dilution of the α-mannosidase suspension to the centrifuge tube, mix well, and place in a constant temperature water bath at 37℃ for 17h to obtain 400μL of sample solution and 400μL of reference solution, respectively.

[0073] (3) Take 400 μL of the sample solution and reference solution 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 30 min. Then add 500 μL of 0.3 mol / L hydrochloric acid, and repeat the extraction with 3 mL of chloroform four times each time. Finally, take the upper aqueous phase and centrifuge it at a speed of 10,000 r / min for 2 min. Collect the supernatant and filter it through a 0.22 μm needle filter to obtain the sample solution and the reference solution.

[0074] (4) Drawing and determination of standard curve:

[0075] Dissolve the mannose standard in 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;

[0076] The standard series working solutions were subjected to derivatization reaction according to step (3);

[0077] The standard series working solutions, test solution and test control solution after the derivatization reaction were measured by high performance liquid chromatography, and the high performance liquid chromatography conditions were:

[0078] Column: Acclaim TM C30, 5 μm 4.6 × 250 mm HPLC column;

[0079] 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;

[0080] Flow rate: 1.0 mL / min;

[0081] Column temperature: 35°C;

[0082] Detector: UV detector;

[0083] Detection wavelength: 254 nm;

[0084] Injection volume: 10 μL;

[0085] The mannose target 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 mannose target peak of the test solution and the test control solution was analyzed to obtain the chromatographic peak area. The concentration corresponding to the mannose chromatographic peak area of ​​the test solution and the test control solution was calculated by the linear regression equation. The content of α-mannan in the yeast culture sample to be tested was calculated according to formula (1)-(3). The calculation formula is:

[0086]

[0087] Where:

[0088] X i -The content of α-mannan in the sample to be tested, expressed as mass percentage, %;

[0089] X i对照 -The content of yeast mannan in the yeast mannan reference substance to be tested, expressed as mass percentage, %;

[0090] c1-Concentration corresponding to the chromatographic peak area of ​​mannose in the test solution, μg / mL;

[0091] c2-Concentration corresponding to the chromatographic peak area of ​​mannose in the test sample control solution, μg / mL;

[0092] m1-the mass of the sample to be tested after hydrolysis, g;

[0093] m2-the mass of the hydrolyzed yeast mannan reference substance, g;

[0094] V1-volume of the sample to be hydrolyzed, mL;

[0095] V2- volume of hydrolyzed yeast mannan reference substance, mL;

[0096] F-Correction factor for low results caused by the destruction or loss of α-mannan in the test sample during the test;

[0097] Purity of P-yeast mannan reference substance, %;

[0098] W- moisture content of yeast mannan reference substance, %;

[0099] n-i.e. 8.5, the volume dilution multiple of the supernatant after collecting the supernatant to prepare the sample solution or the reference solution, adding α-mannosidase, and then adding PMP-methanol solution, sodium hydroxide aqueous solution and hydrochloric acid;

[0100] 0.9 - coefficient for converting mannose content into mannan content;

[0101] X i , X i对照 Repeat the determination with F and X. i The measurement results were expressed as the arithmetic mean ± standard deviation of multiple repeated measurement results, with 3 significant figures retained.

[0102] The specific data of the test are:

[0103] Table 1

[0104]

[0105] Effect test

[0106] 1. Data Statistical Analysis

[0107] GraphPad Prism 9.5.1 was used to analyze and plot the experimental data.

[0108] 2.1 Selection of different enzymatic hydrolysis times

[0109] On the basis of keeping other experimental conditions unchanged, this experiment tested the effect of different enzymatic hydrolysis times (12h, 15h, 16h, 17h, 18h) on the concentration of mannose derivatives in the sample. The experimental results showed (see Figure 1 ): The concentration of mannose derivatives increased significantly with the extension of enzymatic hydrolysis time. At 17h, the concentration of mannose derivatives reached 12.664±0.101 μg / mL, with an RSD of 0.008%; while at 18h, the concentration of mannose derivatives was 12.822±0.124 μg / mL, with an RSD of 0.010%. There was no significant difference between the two (P>0.05), but compared with other enzymatic hydrolysis times, the concentration of mannose derivatives increased significantly (P<0.0001). These results show that the complete hydrolysis time of α-mannosidase to α-mannan in the sample is 17h. Therefore, this experiment determined that the optimal enzymatic hydrolysis time of α-mannosidase is 17h.

[0110] 2.2 Selection of derivation time and extraction times

[0111] Under the premise of keeping other conditions unchanged, this experiment tested the effect of different derivatization reaction times (10min, 30min, 60min) on the concentration of mannose derivatives. The specific experimental data are as follows: at 10min, the concentration was 458.796±8.713μg / mL, RSD was 0.019%; at 30min, the concentration was 477.892±9.105μg / mL, RSD was 0.019%; at 60min, the concentration was 475.181±9.719μg / mL, RSD was 0.020%. The experimental results show (see Figure 3 ), the concentration of mannose derivatives did not increase significantly with the extension of derivatization reaction time. Compared with the reaction time of 10 min, the concentration of mannose derivatives did not increase significantly at 30 min and 60 min (P>0.05). Therefore, in order to improve the reaction efficiency and reduce the time cost, this experiment determined 10 min as the optimal time point for the derivatization reaction.

[0112] In this experiment, dichloromethane was selected as the extraction solvent, and 3 mL was used each time to test the effect of different extraction times (2 times, 3 times, 4 times, and 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.

[0113] 2.3 Linear Relationship

[0114] Under the optimized experimental conditions, the chromatograms of mannose derivatives of mannose standard and experimental group samples are shown in Figure 4 Figure a. By drawing a mannose standard curve Figure 4 In Figure b, we observed that in the concentration range of 1~400 μg / mL, there was a good linear relationship between the mannose concentration and the chromatographic peak area, and the fitting equation was y=79.903x-6.0094, with a correlation coefficient of R²=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 0.063 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 0.208 mg / L. These results show that this method has high precision and accuracy and can be used as a standard method for quantitative analysis.

[0115] 2.4 Method precision and sample repeatability test results

[0116] The results of the method precision test are listed in Table 2. The RSDs were all less than 1%, indicating that the instrument maintained excellent analytical performance under the current chromatographic conditions.

[0117] Table 2 Results of method precision test

[0118]

[0119] As shown in Table 3, the results of the sample repeatability test show that the chromatographic peak area obtained by multiple measurements of the sample does not change much, and the RSD is less than 1%, indicating that the random error of the test is small. Therefore, this method has high reliability and effectiveness.

[0120] Table 3 Sample repeatability test results

[0121]

[0122] 2.5 The test results were summarized and the spike recovery was calculated. The results are shown in Table 4. The samples were spiked in the range of 0.2~0.8 mg, and the average recovery was 88.020%~94.204%, with RSD less than 1%. This shows that the method has high applicability and feasibility and can meet the requirements of routine testing.

[0123] Table 4 Spiked recovery test results

[0124]

[0125] 2.6 Application of the method

[0126] The data in Table 5 show that α-mannan was present in all seven yeast cultures, with the contents ranging from 0.327±0.018% to 0.914±0.009% and RSDs less than 1%.

[0127] Table 5 Detection results of α-mannan in different yeast cultures

[0128]

[0129] Example 2

[0130] Except for the following data, the other steps and parameters of the method are the same as those of Example 1;

[0131] The yeast culture fermentation raw material is the blank group, that is, the dry material state, with a water content of approximately 0%, and the weighed masses are: 0.0791g, 0.0767g, and 0.0806g;

[0132] The mass of yeast mannan standard was weighed: 0.0318 g, with a water content of 0%. After being dissolved in 1.2 mL of ultrapure water, it was divided into three portions, 400 μL each (i.e., 0.0106 g each) and added to the above three blank groups, thus obtaining the following three groups of yeast mannan reference substances;

[0133] The actual masses of yeast mannan reference substances are m2: 0.0897 g, 0.0873 g, and 0.0912 g;

[0134] The theoretical contents of yeast mannan reference substance are: 11.817%, 12.142%, 11.623%; namely, P (100%-W) value;

[0135] Table 6

[0136]

[0137] Example 3

[0138] Except for the following data, the other steps and parameters of the method are the same as those of Example 1;

[0139] The yeast culture fermentation raw material is the blank group, that is, the dry material state, with a water content of approximately 0%, and the weighed masses are: 0.0789g, 0.0802g, and 0.0756g;

[0140] The mass of yeast mannan standard was weighed as follows: 0.0330 g, with a water content of 0%. After being dissolved in 1.2 mL of ultrapure water, it was divided into three portions, 400 μL each (i.e., 0.0110 g yeast mannan standard each) and added to the above three blank groups, thus obtaining the following three groups of yeast mannan reference substances;

[0141] The actual masses of yeast mannan reference substances are m2: 0.0899 g, 0.0912 g, and 0.0866 g;

[0142] The theoretical contents of yeast mannan reference substances are: 12.236%, 12.061%, 12.702%; i.e. P (100%-W) value;

[0143] Table 7

[0144]

[0145] 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 α-mannan in yeast culture, characterized in that: The following steps are involved: (1) Pretreatment: The yeast culture sample to be tested and the yeast mannan reference substance of known purity are crushed and sieved for later use; (2) Hydrolysis: Weigh the pretreated test sample and yeast mannan reference substance of known purity, place them in test tubes, add 12 mol / L glacial sulfuric acid, shake and mix, and immediately place them in ice water for 30 min. Then add water to each test tube, mix and place them in a boiling water bath for 30 min. Transfer the solution in the test tube containing the test sample to a centrifuge tube using a water washing bottle containing MES buffer solution, and transfer the solution in the test tube containing the yeast mannan reference substance to a centrifuge tube using a water washing bottle containing MES buffer solution. conical flask, then add sodium hydroxide aqueous solution to the centrifuge tube and the conical flask respectively, use MES buffer solution to make up to the scale line, adjust the pH to 4.5-5.5 with or without sodium hydroxide aqueous solution, and finally use MES buffer solution to make up to the scale, take the hydrolyzed solution and centrifuge it, collect the supernatant to the bottom of the centrifuge tube, add the dilution of α-mannosidase suspension to the centrifuge tube, mix well, and place it in a constant temperature water bath at 37°C for 17 hours to obtain the sample solution and the reference solution respectively; (3) Derivatization reaction: The sample solution and the reference solution obtained in step (2) were added with PMP-methanol solution and sodium hydroxide aqueous solution, respectively, and heated in a 70°C water bath for 10 min. Hydrochloric acid was then added, and the extraction was repeated 4 times with 3 mL of chloroform each time. Finally, the upper aqueous phase was centrifuged and the supernatant was collected by needle filter to obtain the sample solution and the sample reference solution. (4) Drawing and determination of standard curve: Dissolve the mannose standard in 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, test solution and test control solution after the derivatization reaction were measured by high performance liquid chromatography, and the high performance liquid chromatography conditions were: Column: Acclaim TM C30, 5 μm 4.6 × 250 mm 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 mannose target 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 coordinate and the chromatographic peak area of ​​the standard series working solution as the vertical coordinate to obtain the linear regression equation. The mannose target peak of the test solution and the test control solution was analyzed to obtain the chromatographic peak area. The concentration corresponding to the mannose chromatographic peak area of ​​the test solution and the test control solution was calculated by the linear regression equation. The content of α-mannan in the yeast culture sample to be tested was calculated according to formula (1)-(3). The calculation formula is: Where: X i -The content of α-mannan in the sample to be tested, expressed as mass percentage, %; X i对照 -The content of yeast mannan in the yeast mannan reference substance to be tested, expressed as mass percentage, %; c1-Concentration corresponding to the chromatographic peak area of ​​mannose in the test solution, μg / mL; c2-Concentration corresponding to the chromatographic peak area of ​​mannose in the test sample control solution, μg / mL; m1-the mass of the sample to be tested after hydrolysis, g; m2-the mass of the hydrolyzed yeast mannan reference substance, g; V1-volume of the sample to be hydrolyzed, mL; V2- volume of hydrolyzed yeast mannan reference substance, mL; F-Correction factor for low results caused by the destruction or loss of α-mannan in the test sample during the test; Purity of P-yeast mannan reference substance, %; W-moisture content of yeast mannan reference substance, %; n-i.e. 8.5, the volume dilution multiple of the supernatant after collecting the supernatant to prepare the sample solution or the reference solution, adding α-mannosidase, and then adding PMP-methanol solution, sodium hydroxide aqueous solution and hydrochloric acid; 0.9 - coefficient for converting mannose content into mannan content; X i , X i对照 Repeat the determination with F and X. i The measurement results were expressed as the arithmetic mean ± standard deviation of multiple repeated measurement results, retaining 3 significant figures; The preparation method of the dilution of the α-mannosidase suspension comprises the following steps: Suspend 1 mg of α-mannosidase with an enzyme activity ≥15 U / mg in a mixed aqueous solution of 3 mol / L (NH4)2SO4 and 0.1 mmol / L zinc acetate to obtain 230-250 μL of α-mannosidase suspension with a pH of 7.

5. Add 1 mL of ultrapure water to the α-mannosidase suspension for dilution, store at 4°C, and use within one week.

2. A method for determining the content of α-mannan 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 and a yeast mannan reference substance of known purity are crushed and passed through a 60-mesh sieve for later use.

3. The method for determining the content of α-mannan in yeast culture according to claim 1, characterized in that: In step (2), 0.0600-0.1200 g of the pretreated sample to be tested and 0.0800-0.1100 g of yeast mannan reference substance of known purity are weighed, placed in test tubes, 2.0 mL of glacial sulfuric acid is added, and the mixture is shaken and mixed, and then immediately placed in ice water for incubation. Then 10 mL of water is added to each test tube, and the mixture is mixed and placed in a boiling water bath for incubation. The solution in the test tube containing the sample to be tested is transferred to a 50 mL centrifuge tube using a water washing bottle containing a pH 5.5, 50 mmol / L MES buffer solution. Then 6 mL of an 8 mol / L sodium hydroxide aqueous solution is added to the centrifuge tube, and the volume is adjusted to not more than and close to the 50 mL scale line with a pH 5.5, 50 mmol / L MES buffer solution. The pH is adjusted to 4.5-5.5 with or without a sodium hydroxide aqueous solution. Finally, the volume is adjusted to 50 mL with a pH 5.5, 50 mmol / L MES buffer solution. The solution in the reference test tube was transferred to a 100mL conical flask with a washing bottle containing a pH 5.5, 50mmol / LMES buffer solution, and then 6mL of 8mol / L sodium hydroxide aqueous solution was added to the conical flask, and the pH was adjusted to 4.5-5.5 with or without sodium hydroxide aqueous solution. Finally, the pH was adjusted to 100mL with a pH 5.5, 50mmol / LMES buffer solution. 1-2mL of the hydrolyzed solution was centrifuged at a speed of 13000r / min for 5min, and 0.2mL of the supernatant was collected to the bottom of a 15mL centrifuge tube. 0.2mL of the dilution of the α-mannosidase suspension was added to the centrifuge tube, mixed thoroughly, and placed in a constant temperature water bath to obtain 400μL of sample solution and 400μL of reference solution, respectively.

4. The method for determining the content of α-mannan in yeast culture according to claim 1, characterized in that: In step (3), 400 μL of the sample solution and the reference solution 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 10 min, and then 500 μL of hydrochloric acid with a concentration of 0.3 mol / L is added, and then the extraction is repeated with 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 needle filter to obtain a test solution and a test control solution.

5. A method for determining the content of α-mannan in yeast culture according to claim 3, characterized in that: In step (2), the preparation method of the LMES buffer solution having a pH of 5.5 and a concentration of 50 mmol / L comprises the following steps: Accurately weigh 9.76 g of MES, add it to 900 mL of ultrapure water to dissolve, adjust the pH to 5.5, finally make up to 1 L, and store at 4 °C.

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