CN106950311B - The measuring method of mannan content in a kind of yeast culture - Google Patents
The measuring method of mannan content in a kind of yeast culture Download PDFInfo
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- G—PHYSICS
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Abstract
The measuring method of mannan content in a kind of yeast culture, including following key step: (1) yeast culture, which carries out pretreatment, makes yeast cell wall broken wall therein；(2) reaction is hydrolyzed in pretreated sample in acid condition；(3) after the solution acid alkalinity after sample hydrolysis is tuned into neutrality, reaction is performed the derivatization under certain condition with corresponding derivatization reagent, while mannose standard solution performs the derivatization reaction under the same conditions；(4) using mannose as standard solution, the inverted high performance liquid chromatography of the standard solution after sample and derivatization after derivatization carries out analysis detection；(5) quality of mannose in sample is calculated according to obtained chromatographic data, then is converted into the content of mannosan.The present invention can mannan content in Accurate Determining yeast culture, interfere small, specificity is high.
Description
Technical field
The present invention relates to a kind of measuring methods of mannan content, and in particular to mannosan in a kind of yeast culture
The measuring method of content.
Background technique
Yeast culture is a kind of probiotics, refers under specific process conditions control, passes through on defined medium
Tiny ecosystem product is formed by after abundant anaerobic fermentation.Its main function ingredient is yeast using caused by solid matrix fermentation
Solid matrix, yeast cell contents and yeast cell wall for being denaturalized after extracellular metabolic product, fermentation etc..The product composition
Complexity mainly includes small peptide, organic acid, vitamin, flavour enhancing substance, manna oligosacchride, beta glucan and amino acid and " unknown rush
Growth factor " etc., these substances are the perfect nutritional substrates of microorganism in animal gastrointestinal tract, can be with the life of effective stimulus beneficial bacterium
It is long, their metabolic activity, micro-ecological environment in liptinite are excited, and then improve the production performance of animal.Yeast culture energy
Effect is enough played, wherein yeast cell wall is a very important part, and yeast cells wall construction (Fig. 1) is tough and tensile, main structure
As glucan, mannosan, protein, chitin, lipid etc., as sandwich, outer layer is mannosan, and internal layer is poly- for Portugal
Sugar is sandwiched between one layer of protein molecule, has in the not all saccharomycete of a small amount of component chitin of cell wall, contain
Amount is also because of kind.And mannosan is important one of the polysaccharide component of yeast cell wall, accounts for 40% or so of cell wall dry weight,
Assign activity of cell biology and control cell wall aperture.Mannosan is connected together in the form of covalent bond with protein, by 5%
~20% protein, 80%~90% mannose composition, therefore also referred to as mannan protein, relative molecular weight are
20000~200000, main chain be it is single-stranded, glycosidic bond form is α -1, and 6 connections are connected with branch abundant on main chain, are by sweet dew
Sugar, mannobiose, manninotriose and mannotetrose composition, glycosidic bond form are the connection of α -1,2 or α -1,3.Mannosan is to exempt from
The strongest yeast cell wall polysaccharide of epidemic disease function, it can increase, and animal body fluid is immune and cell immunocompetent, adjusting intestinal flora are flat
Weighing apparatus in conjunction with the exogenous pathogen of absorption, and has the function of anti-radiation, anti-oxidant, antitumor isoreactivity, is in yeast culture
One of important effect ingredient.Therefore, the content of yeast mannans is considered as evaluating a Xiang Chong of yeast culture quality
Want index.
Currently, generally using phend-sulphuric acid and Anthrone-sulfuricacid method to the measurement of mannose content, but use above-mentioned side
It is obtaining the result is that total reduced sugar, can not rule out other sugar when method is used to measure mannan content in yeast culture
The interference of class, specificity is poor, and measurement result not can accurately reflect the actual content of mannosan in sample.In addition, monosaccharide object
Matter can also be used glycan analysis column to be combined Composition distribution detection, but sugared column is not durable, corresponding Composition distribution sensitivity
It is lower, be not suitable for the mannosan in detection yeast culture.And simultaneously because mannosan contains in measurement yeast culture
When amount, yeast cell wall where mannosan is thicker, cannot directly utilize the above method.
In consideration of it, it is an object of the invention to propose the synthesis of a kind of mannan content suitable for yeast culture point
Detection method is analysed, provides technical guarantee for the development and application of yeast culture.
Summary of the invention
The purpose of the present invention is what is be achieved through the following technical solutions, the survey of mannan content in a kind of yeast culture
Determine method, comprising the following steps:
(1) yeast culture is carried out pretreatment makes yeast cell wall broken wall therein；
(2) reaction is hydrolyzed in pretreated sample described in step (1) in acid condition, makes yeast cell wall
Polysaccharide hydrolysis is monosaccharide, and mannosan therein is hydrolyzed into mannose；
(3) after the pH value of solution after hydrolysis described in step (2) being tuned into neutrality, with corresponding derivatization reagent
Reaction is performed the derivatization under certain condition, makes monosaccharide derivatization therein；Then using mannose as standard items, by its standard items
Solution performs the derivatization reaction under the same conditions；
(4) by after derivatization described in step (3) sample solution and the inverted high performance liquid chromatography of standard solution into
Row analysis detection；
(5) quality of mannose in sample is calculated according to obtained chromatographic data in step (4), then is converted into sweet dew
The content of glycan.
Further, in step (1), the yeast cell wall broken wall is using homogenization, ultrasonic treatment or enzymolysis processing.
Further, homogenization is after mixing yeast culture and ultrapure water with the ratio of 1:3~1:7, to use homogenizer
Through 5~12min of homogenization under the conditions of 3000~12000r/min of revolving speed；The ultrasonic treatment is by yeast culture and ultrapure
After water is mixed with the ratio of 1:10~1:20,30~50min of ultrasound under the conditions of 100~150W of ultrasonic power；At the enzymatic hydrolysis
Reason is after mixing yeast culture and ultrapure water with the ratio of 1:4~1:10,1,4 beta-glucanase and glusulase 3:1 in proportion
Addition, additive amount digest pH 4.0~6.0 in 5~13mg/mL, and 40~60 DEG C of hydrolysis temperature, 2~8h of enzymolysis time.
Further, in step (2), reaction is hydrolyzed in sulfuric acid or hydrochloric acid condition.
Further, it is every 20mg yeast culture use that the sulphuric acid hydrolysis, which is according to the use ratio of sample and 72% sulfuric acid,
72% sulfuric acid of 70~100uL stands 1~3h after pressure-resistant glass capsulation pipe sample and sulfuric acid mixing, 5~8 times is added later
The ultrapure water of volume hydrolyzes 3~8h after sealing pressure-resistant glass capsulation pipe at 100 DEG C, uses after hydrolysis completion ice water cooling
6M sodium hydroxide solution is adjusted to neutrality；It is every 20mg ferment that the hydrochloric acid hydrolysis, which is according to the use ratio of sample and 2mol/L hydrochloric acid,
The 2mol/L hydrochloric acid of 2~5mL of female culture, by sample and mixed in hydrochloric acid it is uniform after in seal pipe at 100 DEG C hydrolysis 1~
10h is adjusted to neutrality with 2M sodium hydroxide solution with after ice water cooling after the completion of hydrolysis.
Further, in step (3), the derivatization reagent is 1-phenyl-3-methyl-5-pyrazolones ketone.
Further, the ready-to-use PMP methanol of isometric mannose standard solution or sample solution, 0.5mol/L is molten
Liquid, 0.3mol/L NaOH solution, be placed in tool plug centrifuge tube in, after mixing well in 60~90 DEG C of water-bath derivative reactions 30~
Excessive 0.3mol/L hydrochloric acid solution is added in 90min after letting cool, mix, go out excessive PMP with chloroform extraction, finally will
After the centrifugation of water layer liquid, take supernatant to be measured.
Further, in step (4), the chromatographic condition of the reversed-phase high performance liquid chromatography is with 0.1mol/LpH5.5 acetic acid
Ammonium buffer and acetonitrile form mobile phase according to volume ratio 80/20~60/40 and elute, and flow velocity is 0.5~1.5mL/min, reverse phase
Chromatographic column selects C18 post separation, and UV detector is detected at ultraviolet wavelength 250nm.Every part sample Parallel testing 3 times.
Further, in step (5), the content of mannosan is calculated by formula I in the sample:
In formula I:
X: mannan content %；
A1: in the standard specimen solution after derivative reaction, the average value of mannose peak area；
A2: in the sample solution after derivative reaction, the average value of mannose peak area；
M1: the quality of mannose standard specimen, g；
M2: the quality of yeast culture, g in sample solution；
P: the mass percentage of mannose in mannose standard specimen；
0.9: the conversion coefficient of mannose and mannosan；
Calculated result is accurate to 0.1%.
The present invention makes the abundant broken wall of yeast cells by pretreatment, and without the influence of external ingredient, interference is small, so that sweet dew
Glycan can be fully hydrolyzed in acid condition, then be separated after monosaccharide derivatization using reverse-phase chromatographic column, and select ultraviolet
Detector can more sensitively detect monosaccharide derivatives.
The advantage of the invention is that the content that can accurately measure mannosan in yeast culture is calculated with external standard method,
Interfere small, specificity height and measuring method simplicity, the assay of mannosan suitable for yeast culture and similar sample.
Experiment shows that mannose concentration is in a linear relationship with corresponding peak area within the scope of 1.0~100mg/L in measuring method of the present invention
(R2=0.99), mannosan TIANZHU XINGNAO Capsul is up to 96.3%~102.6%, and relative standard deviation 2.5%, mannosan is most
Low detection limits are up to 0.11mg/L.
Detailed description of the invention
By reading the following detailed description of the preferred embodiment, various other advantages and benefits are common for this field
Technical staff will become clear.The drawings are only for the purpose of illustrating a preferred embodiment, and is not considered as to the present invention
Limitation.And throughout the drawings, the same reference numbers will be used to refer to the same parts.In the accompanying drawings:
Fig. 1 is yeast cell wall structure chart.
Fig. 2 is that mannosan measures process in yeast culture.
Fig. 3 is the HPLC chromatogram of mannose standard solution.
Fig. 4 is the HPLC chromatogram of sample mannose PMP derivative.
Fig. 5 is mannose standard curve.
Specific embodiment
The illustrative embodiments of the disclosure are more fully described below with reference to accompanying drawings.Although showing this public affairs in attached drawing
The illustrative embodiments opened, it being understood, however, that may be realized in various forms the disclosure without the reality that should be illustrated here
The mode of applying is limited.It is to be able to thoroughly understand the disclosure on the contrary, providing these embodiments, and can be by this public affairs
The range opened is fully disclosed to those skilled in the art.
1, instrument
Shimadzu LC-20A high performance liquid chromatograph；High-shear homogenizer (high Buddhist nun AD200L-P)；PB-10 type pH meter (Sai Duoli
This)；Desk centrifuge (Anting Scientific Instrument Factory, Shanghai)；Sonicator (new sesame JY92-IIN).
2, experiment reagent
Methanol (chromatographically pure), acetonitrile (chromatographically pure), chloroform (analysis is pure), hydrochloric acid (analysis is pure), acetic acid (analysis is pure), hydrogen-oxygen
Change sodium (analysis is pure), ammonium acetate (analysis is pure), 1-phenyl-3-methyl-5-pyrazolones ketone (analysis is pure), ultrapure water, mannose mark
Quasi- product (content 98%, Shanghai source leaf biology), mannosan standard items (content 98%, Shanghai source leaf biology), 1,4 beta-glucanase
With glusulase (Hefei Bo Mei biotechnology Co., Ltd).
3, standard solution is prepared
Sweet dew saccharide 0.05g (being accurate to 0.0001g) is accurately weighed in 100mL volumetric flask, it is fixed after being dissolved in water
Hold in 100mL volumetric flask, as Standard Stock solutions.Taken before using 10mL stock solution constant volume into 100mL volumetric flask as
Standard solution.
Embodiment 1
1, the pretreatment of yeast culture sample homogeneous, sulphuric acid hydrolysis
Yeast culture sample 4.5g is accurately weighed in beaker, 30g is added water to and (accurately to 0.0001g), then uses
10min is handled under the conditions of 10 000r/min of high-shear homogenizer；Then the 0.5g in gained homogenizing fluid is accurately weighed as sample,
It is put into 25mL pressure resistance reaction tube, 72% sulfuric acid 2.5mL is added, shakes up 1~3h of standing, after adding ultrapure water 15mL, sealing pressure resistance
Reaction tube stirs hydrolysis 4h at 100 DEG C, and effective ice water cooling will be reacted after the completion of hydrolysis, hydrolyzate is then moved into volumetric flask
In, constant volume to 100mL；Then take wherein 10mL sample solution with the NaOH solution of 6M be adjusted to neutrality, then constant volume is made to 100mL
For testing sample solution.
2, derivatization
Take mannose standard solution or testing sample solution, 0.5mol/L PMP methanol solution (must be ready-to-use),
Each 400 μ L of 0.3mol/L NaOH solution is placed in 10mL tool plug centrifuge tube, in 70 DEG C of water-bath derivative reactions after mixing well
500 μ L of 0.3mol/L hydrochloric acid solution is added in 30min after letting cool, mix, go out excessive PMP with chloroform extraction, uses every time
2.5mL chloroform washs 4 times, finally by after the centrifugation of water layer liquid, takes supernatant to be measured.
3, the measurement of sample
(1) chromatographic condition
Chromatographic columnC18 Bondapak 300mm×4.9mm；Mobile phase: 0.1mol/L ammonium acetate buffer
PH5.5- acetonitrile (V/V, 70:30)；Flow velocity: 1.0mL/min；Column temperature: 35 DEG C；Detection wavelength: 250nm；Sample volume: 20 μ L.
(2) sample measures
Under same chromatographic condition, sample and standard items are injected separately into chromatograph, record the peak face of each chromatographic peak
Product, results are averaged in triplicate for every part of sample.
The content of mannosan is calculated by formula (I) in sample:
In formula I:
X: mannan content %；
A1: in the standard specimen solution after derivative reaction, the average value of mannose peak area；
A2: in the sample solution after derivative reaction, the average value of mannose peak area；
M1: the quality of mannose standard specimen, g；
M2: the quality of yeast culture, g in sample solution；
P: the mass percentage of mannose in mannose standard specimen；
Mannosan measurement process is shown in Fig. 2 in the above yeast culture.
4, analysis of experimental results
(1) chromatographic isolation
With gained mannose and yeast culture HPLC chromatogram under 1 method of embodiment and chromatographic condition, such as Fig. 3,4 institutes
Show, peak 1 indicates the corresponding chromatographic peak of mannose standard solution in Fig. 3, and peak 1 indicates the corresponding chromatographic peak of sample mannose in Fig. 4.
As shown in Figure 4, the separating degree of mannose PMP derivative is greater than 1.5 (as separating degree R=1.5, two component separation degrees can be with
Reach 99.7%, the index that usual R=1.5 is kept completely separate as two adjacent groups point), good separation, baseline is horizontal,
Peak shape is symmetrical.
(2) linear correlation degree and standard curve
According under 1 method of embodiment and chromatographic condition, a series of mannose standard chromatogram of concentration gradients is measured.With
Mannose concentration of standard solution (mg/L) is abscissa x, with corresponding chromatographic peak area (vs) for ordinate y, draws standard
Curve is shown in Fig. 5.
By standard curve (Fig. 5), mannose concentration is in line with corresponding peak area within the scope of 1.0~100mg/L
Sexual intercourse, equation of linear regression y=0.014x+0.006 and related coefficient (R2=0.99).
(3) method precision, TIANZHU XINGNAO Capsul and minimum detection limit
Yeast culture sample is taken, 5 parallel determinations is carried out by above-mentioned processing method and chromatographic condition, measures as a result, simultaneously
Relative standard deviation is found out by statistics.The result shows that the selected yeast culture sample measured by 1 method of embodiment
Mannosan content be 0.80%, relative standard deviation 2.5%.
The yeast culture sample for taking known exact level, is added a certain amount of mannosan standard items, then according to reality
It applies 1 processing method of example and chromatographic condition is measured, reprocess 5 times, measure result with (measured value-background values)/add value
=the rate of recovery, it is 96.3~102.6% that its TIANZHU XINGNAO Capsul, which is calculated,.It is poly- that sweet dew is obtained by weight of 3 times of signal-to-noise ratio pass through
Sugared minimum detection limit (S/N=3) is 0.11mg/L.
Embodiment 2
1, the pretreatment of yeast culture sample ultrasonic, sulphuric acid hydrolysis
Yeast culture sample 4.5g is accurately weighed in beaker, adds water to 30g (accurately to 0.0001g), then super
Ultrasound 35min under the conditions of acoustical power 100W；Then the 0.5g in the ultrasonic liquid of gained is accurately weighed as sample, is put into 25mL pressure resistance
In reaction tube, 72% sulfuric acid 2.5mL is added, shakes up 1~3h of standing, after adding ultrapure water 18mL, seals pressure-resistant reaction tube at 100 DEG C
Lower stirring hydrolyzes 5h, and effective ice water cooling will be reacted after the completion of hydrolysis, is then moved into hydrolyzate in volumetric flask, constant volume arrives
100mL；Then take wherein 10mL sample solution with the NaOH solution of 6M be adjusted to neutrality, then constant volume to 100mL, as sample to be tested
Solution.
2, derivatization
Take mannose standard solution or testing sample solution, 0.5mol/L PMP methanol solution (must be ready-to-use),
Each 400 μ L of 0.3mol/L NaOH solution is placed in 10mL tool plug centrifuge tube, in 75 DEG C of water-bath derivative reactions after mixing well
500 μ L of 0.3mol/L hydrochloric acid solution is added in 35min after letting cool, mix, go out excessive PMP with chloroform extraction, uses every time
2.5mL chloroform washs 4 times, finally by after the centrifugation of water layer liquid, takes supernatant to be measured.
3, the measurement of sample
(1) chromatographic condition
Chromatographic columnC18 Bondapak 300mm×4.9mm；Mobile phase: 0.1mol/L ammonium acetate buffer
PH5.5- acetonitrile (V/V, 80:20)；Flow velocity: 1.1mL/min；Column temperature: 35 DEG C；Detection wavelength: 250nm；Sample volume: 20 μ L.
(2) sample measures
Under same chromatographic condition, sample and standard items are injected separately into chromatograph, record the peak face of each chromatographic peak
Product, every part of sample are repeated twice that results are averaged.
The content of mannosan is calculated by formula (I) in sample:
In formula I:
X: mannan content %；
A1: in the standard specimen solution after derivative reaction, the average value of mannose peak area；
A2: in the sample solution after derivative reaction, the average value of mannose peak area；
M1: the quality of mannose standard specimen, g；
M2: the quality of yeast culture, g in sample solution；
P: the mass percentage of mannose in mannose standard specimen；
The content that the mannosan of yeast culture sample is calculated is 0.86%, relative standard deviation 2.0%, addition
The rate of recovery is 96.6~101.9%.Obtaining mannosan minimum detection limit (S/N=3) by weight of 3 times of signal-to-noise ratio pass through is
0.12mg/L。
Embodiment 3
1, the pretreatment of yeast culture sample enzymatic hydrolysis, hydrochloric acid hydrolysis
Yeast culture sample 4.5g is accurately weighed in beaker, adds water to 30g (accurately to 0.0001g), then by β-
Glucan and papain complex enzyme 3:1 additive amount in proportion, digest pH 6.0, and 60 DEG C of hydrolysis temperature, enzymolysis time 3h；It connects
Accurately weigh gained enzymolysis liquid in 0.5g as sample, be put into 100mL pressure resistance reaction tube, addition 2mol/L hydrochloric acid
50mL seals pressure-resistant reaction tube and stirs hydrolysis 4h at 100 DEG C, it is cold that effective ice water will be reacted after the completion of hydrolysis after mixing
But, then hydrolyzate is moved into volumetric flask, constant volume to 100mL；Then the NaOH solution of wherein 10mL sample solution 6M is taken
Neutrality, then constant volume are adjusted to 100mL, as testing sample solution.
2, derivatization
Take mannose standard solution or testing sample solution, 0.5mol/L PMP methanol solution (must be ready-to-use),
Each 400 μ L of 0.3mol/L NaOH solution is placed in 10mL tool plug centrifuge tube, in 60 DEG C of water-bath derivative reactions after mixing well
500 μ L of 0.3mol/L hydrochloric acid solution is added in 40min after letting cool, mix, go out excessive PMP with chloroform extraction, uses every time
2.5mL chloroform washs 4 times, finally by after the centrifugation of water layer liquid, takes supernatant to be measured.
3, the measurement of sample
(1) chromatographic condition
Chromatographic columnC18 Bondapak 300mm×4.9mm；Mobile phase: 0.1mol/L ammonium acetate buffer
PH5.5- acetonitrile (V/V, 78:22)；Flow velocity: 0.9mL/min；Column temperature: 35 DEG C；Detection wavelength: 250nm；Sample volume: 20 μ L.
(2) sample measures
Under same chromatographic condition, sample and standard items are injected separately into chromatograph, record the peak face of each chromatographic peak
Product, every part of sample are repeated twice that results are averaged.
The content of mannosan is calculated by formula (I) in sample:
In formula I:
X: mannan content %；
A1: in the standard specimen solution after derivative reaction, the average value of mannose peak area；
A2: in the sample solution after derivative reaction, the average value of mannose peak area；
M1: the quality of mannose standard specimen, g；
M2: the quality of yeast culture, g in sample solution；
P: the mass percentage of mannose in mannose standard specimen；
The content that the mannosan of yeast culture sample is calculated is 0.85%, relative standard deviation 3.0%, addition
The rate of recovery is 95.6~102.1%.Obtaining mannosan minimum detection limit (S/N=3) by weight of 3 times of signal-to-noise ratio pass through is
0.10mg/L。
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
In the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of by anyone skilled in the art,
It should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of the claim
Subject to enclosing.
Claims (4)
1. the measuring method of mannan content in a kind of yeast culture, which comprises the following steps:
(1) yeast culture is carried out pretreatment makes yeast cell wall broken wall therein；
(2) reaction is hydrolyzed in pretreated sample described in step (1) in acid condition；
(3) after the pH value of solution after hydrolysis described in step (2) being tuned into neutrality, with corresponding derivatization reagent one
Reaction is performed the derivatization under fixed condition；Then using mannose as standard items, its standard solution is spread out under the same conditions
Biochemical reaction；
(4) by after derivatization described in step (3) sample solution and the inverted high performance liquid chromatography of standard solution divided
Analysis detection；
(5) quality of mannose in sample is calculated according to obtained chromatographic data in step (4), then is converted into mannosan
Content；
Wherein, in step (1), the yeast cell wall broken wall is using enzymolysis processing, and the enzymolysis processing is to train yeast
After object and ultrapure water are supported with the ratio mixing of 1:4~1:10,3:1 is added in proportion for 1,4 beta-glucanase and glusulase, and additive amount is 5
~13mg/mL, digest pH 4.0~6.0,40~60 DEG C of hydrolysis temperature, 2~8h of enzymolysis time；
Wherein, in step (3), the derivatization reagent is 1-phenyl-3-methyl-5-pyrazolones ketone；
Wherein, in step (4), the chromatographic condition of the reversed-phase high performance liquid chromatography is with 0.1mol/L pH5.5 ammonium acetate buffer
Liquid and acetonitrile form mobile phase according to volume ratio 80/20~60/40 and elute, and flow velocity is 0.5~1.5mL/min, reverse-phase chromatographic column
C18 post separation is selected, UV detector is detected at ultraviolet wavelength 250nm.
2. the measuring method of mannan content in yeast culture according to claim 1, which is characterized in that step
(2) in, reaction is hydrolyzed in sulfuric acid or hydrochloric acid condition；It is neutral for adjusting solution with aqueous slkali after the completion of hydrolysis.
3. the measuring method of mannan content in yeast culture according to claim 2, which is characterized in that the sulphur
Sour water solution is 72% sulfuric acid according to the use ratio of sample and 72% sulfuric acid for every 20mg yeast culture with 70~100uL,
1~3h is stood after pressure-resistant glass capsulation pipe sample and sulfuric acid mixing, the ultrapure water of 5~8 times of volumes, sealing pressure resistance are added later
3~8h is hydrolyzed after glass capsulation pipe at 100 DEG C, hydrolysis completion is cooling with ice water, is adjusted to neutrality with 6M sodium hydroxide solution；Institute
State hydrochloric acid hydrolysis be according to the 2mol/L hydrochloric acid of 2~5mL of every 20mg yeast culture, by sample and mixed in hydrochloric acid it is uniform after
1~10h is hydrolyzed at 100 DEG C in seal pipe, is adjusted to neutrality with 2M sodium hydroxide solution with after ice water cooling after the completion of hydrolysis.
4. the measuring method of mannan content in yeast culture according to claim 1, which is characterized in that derivatization
The condition and method of processing are the PMP first that isometric mannose standard solution or sample solution, 0.5mol/L is ready-to-use
Alcoholic solution, 0.3mol/L NaOH solution are placed in tool plug centrifuge tube, in 60~90 DEG C of water-bath derivative reactions after mixing well
Excessive 0.3mol/L hydrochloric acid solution is added in 30~90min after letting cool, mix, go out excessive PMP with chloroform extraction, most
Afterwards by after the centrifugation of water layer liquid, take supernatant to be measured.
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