Method for evaluating content of yeast cell wall polysaccharide in saccharomyces cerevisiae culture

Abstract

The invention discloses a method for evaluating the content of yeast cell wall polysaccharide in a saccharomyces cerevisiae culture, and belongs to the technical field of polysaccharide content determination. The method for evaluating the content of the yeast cell wall polysaccharide in the saccharomyces cerevisiae culture comprises the following steps: S1, sufficiently and uniformly mixing a sample with hydrochloric acid, and performing high-pressure hydrolysis; s2, carrying out derivatization treatment on the hydrolysate by using 5-methyl-2-phenyl-1, 2-dihydropyrazole-3-ketone, and carrying out hydrolysis treatment on the hydrolysate by using the 5-methyl-2-phenyl-1, 2-dihydropyrazole-3-ketone; s3, detecting mannose, glucose and galactose in the derivative sample by using a high performance liquid chromatography-tandem mass spectrometry method; and S4, according to a calibration formula provided by the distribution characteristics of the hydrolyzed mannose and the hydrolyzed galactose in the yeast and the raw materials, obtaining the content of the mannose and the beta-glucan. The method provided by the invention can effectively overcome the interference of polysaccharides from other sources, more accurately evaluate the content of active polysaccharides from the yeast cell wall in the yeast culture, and provide effective technical support for quality control, detection and evaluation of a yeast culture product.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide content detection, and particularly relates to a method for evaluating the polysaccharide content of yeast cell walls in a culture of brewer's yeast. Background Art

[0002] Yeast culture (YC) is a fermented yeast feed ingredient consisting of yeast metabolites, inactivated yeast cells, and fermented nutrients. YC has gained widespread application in animal production due to its benefits in improving animal intestinal health, boosting immunity, replacing antibiotics, and improving meat flavor. Yeast cell wall polysaccharides are a key active component of YC and a crucial indicator of product quality. Direct polysaccharide detection is difficult, but measuring hydrolyzed mannose and glucose provides an effective method for indirectly estimating the content of mannans and β-glucans derived from yeast cell walls in YC. However, hydrolyzed mannose is not solely derived from mannans in the yeast cell wall, leading to biased results. Direct calculation of yeast cell wall β-glucans using hydrolyzed glucose results in significant, unacceptable errors due to interference from glucose released during hydrolysis of the raw material. Therefore, developing more accurate methods for estimating the polysaccharide content of YC based on the detection of hydrolyzed monosaccharides is of practical significance for accurate evaluation of YC product quality control, testing, and application guidance.

[0003] Regarding the detection methods of monosaccharides, the commonly reported methods in recent years include high performance liquid chromatography-differential refractive index detector (HPLC-RID), high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), pre-column derivatization-high performance liquid chromatography-ultraviolet detector (PCD-HPLC-UV), ion chromatography-electrochemical detector (IC-ED), high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), etc. HPLC-RID, HPLC-ELSD, and IC-ED do not require derivatization, but the former two have low sensitivity and selectivity and are susceptible to interference. IC-ED has high sensitivity but requires rigorous cleanup for samples with complex matrices, which increases the difficulty of sample preparation. PCD-HPLC-UV is a more practical method for the determination of monosaccharides, but due to the complex YC matrix, it is time-consuming and lacks selectivity and sensitivity for samples with complex matrices and low target content. Pre-column derivatization (PCD) of monosaccharides followed by HPLC-MS / MS detection overcomes the problem of poor reversed-phase column retention and enhances the ionization efficiency and sensitivity of mass spectrometry. Furthermore, due to the highly diluted sample solution, matrix effects are minimized, facilitating quantitative mass spectrometry. Furthermore, except for sugar isomers that require chromatographic separation, other compounds do not interfere, and detection time is relatively short. However, the application of PCD-HPLC-MS / MS for the detection of hydrolyzed monosaccharides in YC samples combined with effective calibration methods for the evaluation of YC yeast cell wall polysaccharides has not been reported. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a method for evaluating the content of yeast cell wall polysaccharides in a culture of Saccharomyces cerevisiae, which is used to solve the problem of accurately evaluating the content of yeast cell wall polysaccharides in YC.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is to provide a method for evaluating the yeast cell wall polysaccharide content in a culture of Saccharomyces cerevisiae, comprising the following steps:

[0006] S1. Sample hydrolysis: Add concentrated hydrochloric acid to the crushed yeast culture and mix thoroughly. Then dilute with water and hydrolyze under high pressure for 20-60 min. Adjust the pH of the hydrolyzate to neutral, filter, and collect the filtrate.

[0007] S2. Derivatization: Add a derivatizing agent solution and a sodium hydroxide solution to the filtrate, mix thoroughly, and react in a water bath at 50-70°C for 20-60 minutes. Then, add a hydrochloric acid solution for neutralization and mix thoroughly to remove the unreacted derivatizing agent to obtain a derivatized sample.

[0008] S3. performing high performance liquid chromatography-tandem mass spectrometry analysis on the derivatized sample to obtain the contents of hydrolyzed mannose, hydrolyzed glucose, and hydrolyzed galactose;

[0009] S4. Substituting the contents of hydrolyzed mannose and hydrolyzed galactose into a calibration formula proposed based on the distribution characteristics of hydrolyzed mannose and hydrolyzed galactose in yeast and raw materials, the contents of mannan and β-glucan derived from the yeast cell wall are obtained.

[0010] On the basis of the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, the ratio of yeast culture to concentrated hydrochloric acid was 0.5 g:6 mL, and the hydrolysis was carried out in an autoclave at a hydrolysis temperature of 121° C. for 20 min.

[0012] Furthermore, the concentration of the sodium hydroxide solution is 0.3 mol / L, the derivatizing agent solution is a methanol solution containing 5-methyl-2-phenyl-1,2-dihydropyrazol-3-one, and the concentration is 0.5 mol / L; the volume ratio of the filtrate, the sodium hydroxide solution and the derivatizing agent solution is 1:1:1; the temperature of the derivatization reaction is 70°C, and the time is 20 min; the unreacted derivatizing agent is removed with dichloromethane, and the obtained aqueous phase is the derivatized sample.

[0013] Furthermore, the chromatographic conditions are as follows: an Agilent Extend-C18 column with a specification of 3.5 μm 2.1×100 mm, or one with equivalent performance; mobile phase A is an ammonium acetate solution with a concentration of 0.025 mol / L, mobile phase B is acetonitrile, flow rate: 0.3 mL / min, injection volume: 2 μL; column temperature: 35°C; gradient elution program: 0-6.0 min, 25% B; 6.0-6.5 min, 25%-90% B; 6.5-9.5 min, 90% B; post-run 6 min, 25% B; mass spectrometry conditions are: ESI+, multiple reaction monitoring mode detection, precursor ion: m / z 511, fragment ions: m / z 175 and m / z 217.

[0014] Furthermore, the calibration formula is (1) and (2),

[0015] X = (w1 - w2×k1)×0.9 (1),

[0016] Y = X × k2 (2);

[0017] In formula (1) and formula (2), X is the content of mannan, w1 is the content of hydrolyzed mannose, w2 is the content of hydrolyzed galactose, k1 is the ratio of hydrolyzed mannose to galactose in the raw material; Y is the content of glucan, k2 is the ratio of mannose to glucose in the yeast; and 0.9 is the conversion coefficient of mannose to mannan.

[0018] The beneficial effects of the present invention are as follows: for YC samples, the present invention optimizes the hydrolysis conditions, derivatization conditions, and separation and detection conditions, establishes a pre-column derivatization-liquid chromatography-tandem mass spectrometry (PCD-LC-MS / MS) detection method for the three main hydrolyzed monosaccharides of mannose, glucose, and galactose, and proposes a calibration formula for evaluating the yeast cell wall polysaccharide content in YC based on hydrolyzed monosaccharides according to the distribution characteristics of hydrolyzed monosaccharides in yeast cells and raw materials, providing more effective technical support for the quality evaluation, control, and related scientific research of YC products. Under the hydrolysis conditions determined by the present invention, the three hydrolyzed monosaccharides are stable and completely hydrolyzed; within the concentration range examined, the linearity of each hydrolyzed monosaccharide is good, and R 2 The results were all greater than 0.9995; the method spiked recoveries were 93.07% to 105.4%, with RSDs of 1.90% to 6.52%; and the monosaccharide derivatives remained stable over the 96 hours of investigation. Testing of hydrolyzed monosaccharides in actual YC samples and raw materials showed that the content of active yeast cell wall polysaccharides—mannan and β-glucan—in YC can be more accurately assessed based on the ratio of the content of hydrolyzed mannose and glucose in yeast cells and the ratio of the content of hydrolyzed mannose and galactose in the raw materials. The present invention has sufficiently low limits of detection and quantification, and good accuracy, precision, and stability, providing effective technical support for YC product quality control, testing, and evaluation, as well as related scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are the experimental results for optimizing hydrolysis conditions; A represents the optimization of hydrochloric acid dosage, B represents the optimization of autoclave hydrolysis time, and C represents the optimization of pressure-resistant hydrolysis tube time.

[0020] Figure 2 The effect of derivatization conditions on the yield of three monosaccharide derivatives; A is the optimization of derivatization temperature, B is the optimization of derivatization time, and C is the optimization of derivatization agent dosage;

[0021] Figure 3 The MRM chromatograms of three sugar derivatives in the standard and sample are shown in Figure 1. The peaks in the figure from left to right are mannose, glucose, and galactose.

[0022] Figure 4 The stability of three hydrolyzed polysaccharide derivatives; A is the stability of the monosaccharide derivative in the standard and sample matrices; B is the stability of the monosaccharide derivative after 10-fold dilution with pure water after derivatization. DETAILED DESCRIPTION

[0023] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.

[0024] Example 1

[0025] 1. Materials and Reagents

[0026] Saccharomyces cerevisiae culture was provided by Beijing Zhongnong Hongke Biotechnology Co., Ltd.; D-(+)-mannose, D(+)-anhydroglucose, D-galactose, and mannan, purity ≥98%, were provided by Shanghai Aladdin Biochemical Technology Co., Ltd.; 5-methyl-2-phenyl-1,2-dihydropyrazol-3-one (PMP), purity ≥99.5%, was provided by Shanghai MacLean Biochemical Technology Co., Ltd.; ammonium acetate and acetonitrile, chromatographic grade, were provided by Tianjin Biaoshiqi Technology Development Co., Ltd.; other reagents were of analytical grade unless otherwise specified; ultrapure water was used in the laboratory.

[0027] 2. Instruments and Equipment

[0028] Agilent 6410 Triple Quad LC / MS liquid chromatography-tandem mass spectrometer, Agilent Technologies, USA; TOMY SX-500 autoclave, SANYO Co., Ltd., Japan; Centrifuge 5810R centrifuge, Eppendorf GmbH, Germany; KQ-500DE CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; HH-S digital constant temperature water bath, Changzhou Xiangtian Testing Instrument Factory; MS 3basic vortex mixer, IKA Group, Germany; NW30VF Smart Plus ultrapure water machine, Likang Biomedical Technology Holdings Co., Ltd.; pressure-resistant hydrolysis tubing, Yancheng Pengyu Scientific Research Glass Instrument Co., Ltd.

[0029] 3. Solution Preparation

[0030] (1) Standard stock solution: Weigh 50 mg (accurate to 0.01 mg) of mannose, glucose, galactose, and mannan, respectively, dissolve them in water and dilute to volume in a 50 mL volumetric flask, shake well, and obtain a stock solution of each standard with a concentration of 1 mg / mL. Store the solution in a storage bottle at 4°C and bring to room temperature before use.

[0031] (2) Series of mixed standard working solutions: Pipette 50.0, 100.0, 200.0, 300.0, 400.0, and 500.0 μL of mannose and galactose stock solutions and 0.50, 1.00, 2.00, 3.00, 4.00, and 5.00 mL of glucose stock solution, respectively, and dilute to the volume with water into a series of 10 mL volumetric flasks to obtain a series of mixed standard working solutions with mannose and galactose concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, and glucose concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL.

[0032] (3) Derivatizing agent solution: Weigh 4.35 g (accurate to 1 mg) of 5-methyl-2-phenyl-1,2-dihydropyrazol-3-one (PMP), dissolve it in methanol, and dilute to 50 mL to obtain a derivatizing agent solution with a PMP concentration of 0.5 mol / L.

[0033] (4) Sodium hydroxide solution: Weigh 120 g of sodium hydroxide and slowly add 200 mL of water into a beaker to dissolve it completely. After it is completely cooled, transfer it to a 500 mL volumetric flask and adjust the volume to obtain a sodium hydroxide solution with a concentration of 6 mol / L. Dilute it 20 times with water to obtain a sodium hydroxide solution with a concentration of 0.3 mol / L.

[0034] (5) Hydrochloric acid solution: Pipette 12.5 mL of concentrated hydrochloric acid and slowly add 200 mL of water into a beaker to mix thoroughly. After cooling completely, transfer to a 500 mL volumetric flask and adjust the volume to obtain a hydrochloric acid solution with a concentration of 0.3 mol / L.

[0035] (6) Ammonium acetate solution: Weigh 1.5416 g of ammonium acetate, add appropriate amount of water to dissolve, dilute to 1000 mL, and filter through a 0.22 μm filter to obtain an ammonium acetate solution with a concentration of 0.02 mol / L.

[0036] Example 2

[0037] 1. Sample Pretreatment

[0038] (1) Sample hydrolysis: Accurately weigh 0.5 g (accurate to 0.1 mg) of the crushed sample into a 150 mL conical flask, add 6 mL of concentrated hydrochloric acid, mix thoroughly, add 44 mL of water, plug the flask, and place in an autoclave at 121°C for 20 min. Remove and cool to room temperature. Use 6 mol / L and then 0.3 mol / L sodium hydroxide solutions, combined with precision pH test paper, to adjust the pH of the hydrolyzate to neutral. Then transfer the hydrolyzate to a 100 mL volumetric flask and make up to volume with water. Take an appropriate amount of the hydrolyzate and filter it. The filtrate is set aside.

[0039] (2) Sample derivatization: Pipette 200 μL of hydrolyzate into a 15 mL plastic centrifuge tube, add 400 μL of derivatizing agent solution and 400 μL of 0.3 mol / L sodium hydroxide solution, mix thoroughly, and derivatize in a 70°C water bath for 60 min. After cooling to room temperature, add 500 μL of 0.3 mol / L hydrochloric acid solution and mix thoroughly. Add 5 mL of dichloromethane, vortex for 1 min, centrifuge at 3000 r / min for 5 min, remove the lower dichloromethane phase, and extract the upper aqueous phase with dichloromethane three times to remove excess derivatizing agent, and filter through a 0.22 μm filter membrane. Derivatize a series of standard solutions using the same method, and detect the derivatized samples and standards on a liquid chromatograph.

[0040] 2. Analysis conditions

[0041] (1) Chromatographic conditions: Agilent Extend-C18 (3.5 μm, 2.1 × 100 mm). Mobile phase A was 0.025 mol / L ammonium acetate solution, mobile phase B was acetonitrile, flow rate: 0.3 mL / min, injection volume: 2 μL; column temperature: 35°C. Gradient elution program: 0–6.0 min, 25% B; 6.0–6.5 min, 25%–90% B; 6.5–9.5 min, 90% B; post-run: 6 min, 25% B.

[0042] (2) Mass spectrometry conditions: ESI+ ion source, capillary voltage 4 kV, drying gas flow rate 12 L / min, drying gas temperature 350°C, nebulizer gas pressure 20 psi, ion source temperature 150°C, and quadrupole temperature 100°C. Multiple reaction monitoring (MRM) parameters are shown in Table 1.

[0043] Table 1 MRM parameters of three hydrolyzed monosaccharides

[0044]

[0045] Note: * indicates quantitative ion.

[0046] Example 3

[0047] Optimization of sample pretreatment conditions

[0048] (1) Optimization of hydrolysis conditions: Take 0.5 g of yeast culture, control the total hydrolyzate volume to 50 mL, and control the hydrolysis time to 60 min; ① Use 2, 4, 6, 8, and 10 mL of concentrated hydrochloric acid, respectively, and other conditions are the same as the hydrolysis steps of the sample in Example 2, and optimize the amount of hydrochloric acid; ② Fix the amount of hydrochloric acid and optimize the hydrolysis time under the conditions of 20, 40, 60, 80, and 100 min; ③ Fix the amount of hydrochloric acid and use a pressure-resistant hydrolysis tube to observe the hydrolysis conditions under the conditions of 20, 40, 60, 80, and 100 min.

[0049] For YC samples, the applicability of hydrolysis in an autoclave (hydrolysis temperature of 121°C) with different concentrations of hydrochloric acid was optimized, such as Figure 1 The effect of hydrochloric acid dosage shown in A shows that with the increase of hydrochloric acid concentration, the response values of the three monosaccharide derivatives have a downward trend, among which the downward trend of glucose is the most obvious. This is due to the degradation of monosaccharides by high concentration of hydrochloric acid. Considering that mannose is an important indicator, the hydrochloric acid dosage is selected as 6mL. The experiment further optimized the hydrolysis time, such as Figure 1The results of B show the effect of hydrolysis time in autoclave. The results show that the product content remains stable when the hydrolysis time is 20 to 60 minutes, so the shortest hydrolysis time of 20 minutes is selected. Considering the versatility of the method, the experiment further compares the hydrolysis results in a pressure-resistant glass water bath (98°C) with the same amount of hydrochloric acid. Figure 1 The influence of hydrolysis time on the pressure-resistant hydrolysis tube shown in C is that at least 60 minutes of hydrolysis is required to achieve the same hydrolysis effect as that of a high-pressure sterilizer.

[0050] (2) Optimization of derivatization conditions: 100 μL of each of 1 mg / mL mannose, glucose, and galactose standard solutions were used to control the total reaction volume to 1 mL. If the volume was insufficient, pure water was added. The rest of the derivatization steps were carried out according to the sample derivatization steps in Example 2. 4 ) orthogonal table, and a three-factor, three-level orthogonal experiment was conducted to optimize the temperature, time, and derivatization agent concentration. Temperatures: 50°C, 70°C, and 90°C; time: 20, 40, and 60 min; and derivatization agent concentration: 0.1, 0.2, and 0.3 mol / L, respectively, by adding 200, 400, and 600 μL of a 0.5 mol / L PMP solution.

[0051] Under alkaline conditions, two PMP molecules react with one monosaccharide molecule to produce a sugar derivative with UV absorption. Orthogonal experiments were conducted on temperature, time, and concentration of the derivatizing agent. The results are as follows: Figure 2 The results show that the derivatization agent has the greatest impact, followed by temperature, and the derivatization time has the least impact. The optimal derivatization agent concentration in the system is 0.2 mol / L, at which the PMP concentration is approximately 90 times the monosaccharide concentration. High temperatures reduce the galactose response, so 70°C is recommended. Reaction time has little impact and can be adjusted to 20 to 60 minutes depending on the specific situation.

[0052] (3) Optimization of detection conditions

[0053] In this experiment, an acetonitrile-ammonium acetate mobile phase system was used. Under conditions of relatively low ammonium acetate concentration, the binary gradient elution conditions were optimized. Since the three monosaccharides are isomers, they cannot be distinguished by mass spectrometry and must be separated by chromatography. Under the optimized elution conditions (i.e., the chromatographic conditions in Example 2), the three sugar derivatives in the sample were satisfactorily separated, and the residual PMP and matrix did not interfere with the determination. Figure 3 shown.

[0054] Example 4

[0055] Methodological evaluation

[0056] (1) Linearity, detection limit, and quantification limit: The chromatographic peak areas of the series of standard solutions after derivatization were regressed with the corresponding monosaccharide concentrations to establish a standard curve, and the linearity was evaluated by the correlation coefficient. With reference to the blank sample, the detection limit and quantification limit of the method were calculated with the signal-to-noise ratio of the chromatographic peak of the monosaccharide derivative being not less than 3 times and less than 10 times, respectively.

[0057] According to the possible range of the three hydrolyzed monosaccharides in the sample hydrolyzate, the concentration range shown in Table 2 was investigated, and the method showed excellent linearity, R 2 Under the sample pretreatment component of the present invention, the detection limit of the three monosaccharides is less than or equal to 0.003 mg / g, and the quantification limit is less than or equal to 0.01 mg / g, which can meet the detection requirements of low target content in complex matrices.

[0058] Table 2 Linearity, detection limit and quantification limit of three hydrolyzed monosaccharides

[0059]

[0060] Note: y is the peak area of sugar derivatives, x is the concentration (μg / mL)

[0061] (2) Accuracy, precision and stability of derivative products: Six repetitions of monosaccharide spike recovery tests with 2 to 3 levels of target content in actual samples were performed to evaluate the accuracy and precision of the hydrolysis process; polysaccharides were added to actual samples to evaluate the effect of the sample matrix on the hydrolysis of polysaccharides during the hydrolysis process and to evaluate the reliability of the method; the derivatized sample solution and the diluted sample solution after derivatization were continuously injected within 96 hours, and the stability of the derivative product was evaluated by the change in the response value.

[0062] The completeness of polysaccharide hydrolysis in the sample matrix and the hydrolytic stability of the three monosaccharides affect the accuracy and precision of the determination. In this experiment, samples spiked with mannan and the three target monosaccharides were hydrolyzed. The accuracy and precision of the method were evaluated by spiked recoveries and relative standard deviations. The completeness of polysaccharide hydrolysis and the stability of the target monosaccharides during the hydrolysis process were also examined. The results are shown in Table 3. The spiked recoveries of the three monosaccharides ranged from 93.07% to 105.4%, with RSDs of 1.90% to 6.52%, indicating good accuracy and precision. This also indicates that the three hydrolyzed polysaccharides remained stable during the hydrolysis process. The recovery of mannan ranged from 100.5% to 101.4%, with RSDs of 0.97% to 1.94%, indicating good accuracy and precision for mannan and that the hydrolysis of mannan to mannose was relatively complete under the experimental conditions.

[0063] Table 3 Accuracy and precision of detection of three hydrolyzed monosaccharides

[0064]

[0065] After the sample solution is hydrolyzed and derivatized, the stability of the monosaccharide derivatives in the sample solution will have a great influence on the determination results. This experiment continuously monitors the peak area changes of the three hydrolyzed monosaccharides in the standard sample and sample solution after derivatization for 96 hours, as well as the peak area changes of the three hydrolyzed monosaccharides in the standard sample and sample solution diluted 10 times after derivatization. Figure 4 The results show that the monosaccharide derivatives remain stable in both the standard and sample matrices, and that dilution of the derivatized sample has no effect on stability. Therefore, after derivatization, if the concentration of the sample exceeds the linear range, it is not necessary to dilute the hydrolyzate and re-derivate it; the derivatized sample can be directly diluted to simplify the experimental procedure.

[0066] Example 5

[0067] Determination of hydrolyzed monosaccharides in actual YC samples and evaluation of the mannan and β-glucan content of the source yeast

[0068] This method was applied to nine commercially available YC products, as well as raw materials such as soybean meal, bran, and yeast. The results are shown in Table 4. The mannose-to-glucose ratio in yeast was 1:1.46, consistent with the literature. Since it lacks galactose, this suggests that the hydrolyzed galactose in YC originates from the raw materials. Common YC production raw materials, soybean meal, soybean cake, and bran, contain three hydrolyzed monosaccharides. The mannose-to-galactose ratio in soybean meal and soybean cake is close to 1:10, while in bran, it is close to 1:3. Based on the distribution characteristics of the three hydrolyzed monosaccharides in these YC products and raw materials, using hydrolyzed mannose to directly calculate the content of mannan, an active polysaccharide in the yeast cell wall, results in significant errors. However, since glucose is more widely available, such as starch, cellulose, and soy oligosaccharides, which can be hydrolyzed to release glucose, using hydrolyzed glucose content to directly calculate β-glucan, an active polysaccharide in the yeast cell wall, results in unacceptable errors. Therefore, the contents of mannan and glucan in YC can be corrected according to formula (1) and formula (2).

[0069] X = (w1 - w2 × k1) × 0.9 (1)

[0070] Y = X × k2 (2)

[0071] In formulas (1) and (2), X is the mannan content, expressed in mg / g; w1 is the hydrolyzed mannose content, expressed in mg / g; w2 is the hydrolyzed galactose content, expressed in mg / g; k1 is the ratio of hydrolyzed mannose to galactose in the raw material; Y is the glucan content, expressed in mg / g; k2 is the ratio of mannose to glucose in the yeast; and 0.9 is the conversion coefficient for mannose to mannan. Taking soybean meal-based YC as an example, k1 = 0.1 and k2 = 1.46. The mannan and β-glucan contents derived from yeast cell walls calculated according to formulas (1) and (2) are also listed in Table 4.

[0072] Table 4 Detection results of three hydrolyzed monosaccharides in brewing YC

[0073]

[0074]

[0075] Note: ND means not detected; the YC, k1 and k2 values of different raw materials are different and are subject to actual measurement.

[0076] The present invention establishes a method based on high-pressure rapid hydrolysis-liquid chromatography-tandem mass spectrometry to efficiently and selectively detect hydrolyzed mannose, hydrolyzed glucose, and hydrolyzed galactose in YC. Combined with the distribution characteristics of hydrolyzed mannose and hydrolyzed galactose, the content of active polysaccharides from yeast cell walls in YC can be more accurately assessed, providing more effective technical support for the quality monitoring, evaluation, and application of YC products.

[0077] In the present invention, data acquisition, qualitative and quantitative calculations were performed using MassHunter Workstation Software Wersion B.09.00; data processing and analysis of the results were performed using WPS Office 2024 and Origin 2024.

[0078] Although the specific embodiments of the present invention have been described in detail in conjunction with the embodiments, this should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for evaluating the content of yeast cell wall polysaccharides in a culture of Saccharomyces cerevisiae, characterized in that: The following steps are involved: S1. Sample hydrolysis: Add concentrated hydrochloric acid to the crushed yeast culture and mix thoroughly. Then dilute with water and hydrolyze under high pressure for 20-60 min. Adjust the pH of the hydrolyzate to neutral, filter, and collect the filtrate. S2, derivatization: adding a derivatizing agent solution and a sodium hydroxide solution to the filtrate, mixing thoroughly, and reacting in a water bath at 50-70° C. for 20-60 minutes, then adding a hydrochloric acid solution for neutralization, mixing thoroughly, and removing unreacted derivatizing agent to obtain a derivatized sample; S3. performing high performance liquid chromatography-tandem mass spectrometry analysis on the derivatized sample to obtain the contents of hydrolyzed mannose, hydrolyzed glucose, and hydrolyzed galactose; S4. Substituting the contents of hydrolyzed mannose and hydrolyzed galactose into a calibration formula proposed based on the distribution characteristics of hydrolyzed mannose and hydrolyzed galactose in yeast and raw materials, the contents of mannan and β-glucan derived from the yeast cell wall are obtained.

2. The method for evaluating the yeast cell wall polysaccharide content in a culture of Saccharomyces cerevisiae according to claim 1, wherein: The ratio of yeast culture to concentrated hydrochloric acid was 0.5 g:6 mL, and the hydrolysis was carried out in an autoclave at a temperature of 121° C. for 20 min.

3. The method for evaluating the yeast cell wall polysaccharide content in a culture of Saccharomyces cerevisiae according to claim 1, wherein: The concentration of the sodium hydroxide solution is 0.3 mol / L, and the derivatizing agent solution is a methanol solution containing 5-methyl-2-phenyl-1,2-dihydropyrazol-3-one with a concentration of 0.5 mol / L. The volume ratio of the filtrate, the sodium hydroxide solution, and the derivatizing agent solution is 1:2:

2. The temperature of the derivatization reaction is 70° C., and the time is 20 minutes. Unreacted derivatizing agent is removed with dichloromethane, and the resulting aqueous phase is the derivatized sample.

4. The method for evaluating the content of yeast cell wall polysaccharides in a culture of Saccharomyces cerevisiae according to claim 1, wherein: Chromatographic conditions: An Agilent Extend-C18 column with specifications of 3.5 μm 2.1×100 mm was used; mobile phase A was 0.025 mol / L ammonium acetate solution, mobile phase B was acetonitrile, flow rate: 0.3 mL / min, injection volume: 2 μL; column temperature: 35°C; gradient elution program: 0-6.0 min, 25% B; 6.0-6.5 min, 25%-90% B; 6.5-9.5 min, 90% B; post-run 6 min, 25% B; mass spectrometry conditions: ESI+, multiple reaction monitoring mode detection, precursor ion: m / z 511, fragment ions: m / z 175 and m / z 217.

5. The method for evaluating the yeast cell wall polysaccharide content in a culture of Saccharomyces cerevisiae according to claim 1, wherein: The calibration formulas are (1) and (2), X = (w1 - w2×k1)×0.9 (1), Y = X × k2 (2); In formula (1) and formula (2), X is the content of mannan, w1 is the content of hydrolyzed mannose, w2 is the content of hydrolyzed galactose, k1 is the ratio of hydrolyzed mannose to galactose in the raw material; Y is the content of β-glucan, k2 is the ratio of hydrolyzed mannose to hydrolyzed glucose in the yeast; and 0.9 is the conversion coefficient of mannose to mannan.

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