CN105628457B - The extracting method and content assaying method of yeast beta-dextran in a kind of liquid milk - Google Patents

The extracting method and content assaying method of yeast beta-dextran in a kind of liquid milk Download PDF

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CN105628457B
CN105628457B CN201410579236.5A CN201410579236A CN105628457B CN 105628457 B CN105628457 B CN 105628457B CN 201410579236 A CN201410579236 A CN 201410579236A CN 105628457 B CN105628457 B CN 105628457B
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dextran
liquid
yeast beta
liquid milk
beta
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CN105628457A (en
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侯慧敏
陈伟
陈云
刘宇
史玉东
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Inner Mongolia Mengniu Dairy Group Co Ltd
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Inner Mongolia Mengniu Dairy Group Co Ltd
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Abstract

The present invention provides a kind of extracting method of yeast beta glucan in liquid milk, including：A, 25 grams of liquid milks are taken, 0.01 0.05U of 0.03 0.1U of alkali protease and neutral proteinase is added, mixes when 40 60 DEG C of heating water baths 24 are small, obtain hydrolyzate；B, mix and centrifuged 15 20 minutes after 5,000 6000 revs/min, remove supernatant, retain sediment and upper-layer fat；C, 15 30mL of deionized water is added into sediment and upper-layer fat, mixes and is centrifuged 15 20 minutes after 5,000 6000 revs/min, removes supernatant and upper-layer fat, retains precipitation；D, precipitation is washed with water one to twice, obtains extract.The extracting method effectively reduces the loss of beta glucan in extraction process；Eliminate the lactose of interference measurement.Present invention also offers the assay method using yeast beta glucan in the liquid milk of the extracting method.

Description

The extracting method and content assaying method of yeast beta-dextran in a kind of liquid milk
Technical field
The present invention relates to the extracting method and content assaying method of yeast beta-dextran in a kind of food, liquid is particularly suitable for The extraction of yeast beta-dextran and measure in state breast.
Background technology
Yeast beta-dextran be using saccharomyces cerevisiae as raw material, the processing of extracted, soda acid, spray drying and etc. production and Into molecular weight is 20,000 to 4,000,000 dalton, and molecular formula is (C6H12O6)n, wherein n=125-25000.Numerous studies prove, ferment Female beta glucan has immune enhancing, radioresistance and hypolipemic function, this causes yeast beta-dextran to have more in food service industry To be widely applied prospect.
At present, the assay method of yeast beta-dextran uses phend-sulphuric acid more, but the method precision is poor, and not The yeast beta-dextran being adapted in measure liquid diary product.The comparison of ingredients of liquid diary product is complicated, and contains a large amount of protein, The result accuracy directly measured is poor and can cause harmful effect to instrument.Ideal scheme is first to extract in liquid milk Yeast beta-dextran, then the content of yeast beta-dextran is measured.Existing extracting method is to use protein precipitant（Example Such as zinc acetate and potassium ferrocyanide）By the protein precipitation in liquid milk, supernatant is taken to be measured；The shortcomings that this method is： The rate of recovery for easily causing beta glucan is low；In addition, in salt precipitation protein process, lactose and other more sugar additives are to ion color Spectrum measure has interference, influences the measure of beta glucan.
The content of the invention
The object of the present invention is to provide a kind of extracting method of yeast beta-dextran in liquid milk, can reduce in extraction process The loss of yeast beta-dextran.
It is a further object to provide a kind of content assaying method of yeast beta-dextran in liquid milk, it is recycled Rate and precision are higher.
The present invention provides a kind of extracting method of yeast beta-dextran in liquid milk, including：A, 25 grams of liquid milks are taken, are added Enter alkali protease 0.03 to 0.1U and neutral proteinase 0.01 to 0.05U, fully vibrate mixing and add after 40 to 60 DEG C of water-baths When heat 2 to 4 is small, hydrolyzate is obtained；B, by hydrolyzate vibration after mixing, 15 to 20 are centrifuged in 5000 to 6000 revs/min Minute, remove supernatant, retain sediment and upper-layer fat；C, to the obtained sediments of step b and 60 are added in upper-layer fat After mixing, centrifuge 15 to 20 minutes, go in 5000 to 6000 revs/min to 100 DEG C of deionized water 15 to 30mL, vibration Fall supernatant and upper-layer fat, retain precipitation；And d, the precipitation obtained in step c washed with water one to twice, carried Take thing；The method washed with water is：60 to 100 DEG C of deionized water 15 is added to 30mL, vibration after mixing, in 5000 to 6000 revs/min centrifuge 15 to 20 minutes, remove supernatant, retain precipitation.
In liquid milk in a kind of exemplary embodiment of the extracting method of yeast beta-dextran, further include step d Obtained extract freeze-drying.
Present invention also offers a kind of content assaying method of yeast beta-dextran in liquid milk, including first pass through above-mentioned The extracting method of yeast beta-dextran in liquid milk, extracts the yeast beta-dextran in liquid milk to be measured, then measures extraction product The content of middle yeast beta-dextran.
In liquid milk in a kind of exemplary embodiment of the content assaying method of yeast beta-dextran, assay method bag Include：By above-mentioned extracting method, the yeast beta-dextran in liquid milk to be measured is extracted；By the product obtained after extraction with molten wall Enzyme, 1,6- dextranases, circumscribed -1,4 beta-glucanase and beta-glucosidase enzyme hydrolysis, make yeast beta-dextran all be hydrolyzed into Portugal Grape sugar, obtains prepare liquid；And the content of glucose in prepare liquid is measured, and calculate yeast beta-dextran in liquid milk to be measured Content.
In liquid milk in a kind of exemplary embodiment of the content assaying method of yeast beta-dextran, prepare liquid is measured The method of the content of middle glucose is GOPOD methods.
In liquid milk in a kind of exemplary embodiment of the content assaying method of yeast beta-dextran, GOPOD methods Step includes：3 mL GOPOD solution are added into prepare liquid, are kept for 20 minutes in 40 DEG C of water-baths；It is finally cooled to room temperature simultaneously Absorbance is measured under 510nm.
In liquid milk in a kind of exemplary embodiment of the content assaying method of yeast beta-dextran, prepare liquid is measured The method of the content of middle glucose is high performance liquid chromatography.
In liquid milk in a kind of exemplary embodiment of the content assaying method of yeast beta-dextran, high-efficient liquid phase color The condition of spectrometry is：Chromatographic column：4 × 50 mm of 4 × 250 mm of Dionex CarboPac PA10, band CarboPac PA10 Guard column；Mobile phase：A is ultra-pure water, resistivity >=18.2M Ω；B is 0.25 mol/L sodium hydroxide solutions；C is 1 mol/L Sodium acetate solution；Detector：Ampere detector；Au working electrodes；Ag/AgCl reference electrodes；30 DEG C of detection cell temperature；Sugar is adopted With Integrated amperometry mode detection, four current potential of waveform selection carbohydrate standard；Flow velocity：1.0 mL/min；Column temperature：30 ℃；Into Sample amount：25 μL；
Elution gradient see the table below：
Time, min Flow velocity, mL/min OH concentration, mmol/L Sodium acetate concentration, mol/L Gradient curve
0.00 1.0 15 0 Linearly
16.00 1.0 15 0 Linearly
16.10 1.0 100 0.6 Linearly
21.00 1.0 100 0.6 Linearly
21.10 1.0 200 0 Linearly
23.00 1.0 200 0 Linearly
23.10 1.0 15 0 Linearly
30.00 1.0 15 0 Linearly
The sugared waveform parameter of ampere detector detection：
Time, s Voltage, V It is integrated
0.000 0.100 off
0.200 0.100 on
0.400 0.100 off
0.410 -2.000 off
0.420 -2.000 off
0.430 0.600 off
0.440 -0.100 off
0.500 -0.100 Off, LastStep on
The extracting method of yeast beta-dextran in liquid milk provided by the invention, is removed in liquid milk using protease hydrolytic Protein, effectively reduce loss of the yeast beta-dextran in extraction process；And eliminated in extraction process to surveying Fixed noisy lactose.Yeast beta-dextran in liquid milk is measured using the extracting method, measures the rate of recovery and precision Degree is higher.
Embodiment
In order to which the technical features, objects and effects of invention are more clearly understood, illustrate in conjunction with following embodiments The embodiment of the present invention.
" U ", i.e. unit of activity in the present invention, in optimum condition（25℃）Under, 1 micromole of interior catalysis per minute（μmol）Bottom The enzyme amount that thing is converted into needed for product is set to a unit of activity.
Extract example 1：The extracting method of yeast beta-dextran in liquid milk.
A, take 25 grams of liquid milks, add alkali protease 0.04 U and neutral proteinase 0.03U, fully vibration mixing after When 40 DEG C of heating water baths 2 are small, hydrolyzate is obtained；
B, by hydrolyzate vibration after mixing, centrifuged 15 minutes in 5000 revs/min, remove supernatant, retain precipitation Thing and upper-layer fat；
C, 60 DEG C of 15 mL of deionized water is added into the obtained sediments of step b and upper-layer fat, vibration is uniformly mixed Afterwards, centrifuged 15 minutes in 5000 revs/min, remove supernatant and upper-layer fat, retain precipitation；
D, the precipitation obtained in step c is washed with water one to twice, obtains extract；The method washed with water is：Add Enter 60 DEG C of 15 mL of deionized water, vibration after mixing, centrifuges 15 minutes in 5000 revs/min, removes supernatant, retains Precipitation；
E, the extract for obtaining step d is freeze-dried, and the temperature of freeze-drying is -47 DEG C, extract after being dried.
Extract example 2：The extracting method of yeast beta-dextran in liquid milk.
A, take 25 grams of liquid milks, add 0.01 U of alkali protease 0.03U and neutral proteinase, fully vibration mixing after When 55 DEG C of heating water baths 2.5 are small, hydrolyzate is obtained；
B, by hydrolyzate vibration after mixing, centrifuged 17 minutes in 5500 revs/min, remove supernatant, retain precipitation Thing and upper-layer fat；
C, 80 DEG C of 20 mL of deionized water is added into the obtained sediments of step b and upper-layer fat, vibration is uniformly mixed Afterwards, centrifuged 17 minutes in 5500 revs/min, remove supernatant and upper-layer fat, retain precipitation；
D, the precipitation obtained in step c is washed with water one to twice, obtains extract；The method washed with water is：Add Enter 80 DEG C of deionized water 20mL, vibration after mixing, centrifuges 17 minutes in 5500 revs/min, removes supernatant, and it is heavy to retain Form sediment；
E, the extract for obtaining step d is freeze-dried, and the temperature of freeze-drying is -47 DEG C, extract after being dried.
Extract example 3：The extracting method of yeast beta-dextran in liquid milk.
A, 25 grams of liquid milks are taken, add alkali protease 0.1U and neutral proteinase 0.05U, fully vibration mixing is after 60 When DEG C heating water bath 4 is small, hydrolyzate is obtained；
B, by hydrolyzate vibration after mixing, centrifuged 20 minutes in 6000 revs/min, remove supernatant, retain precipitation Thing and upper-layer fat；
C, 100 DEG C of 30 mL of deionized water is added into the obtained sediments of step b and upper-layer fat, vibration mixing is equal After even, centrifuged 20 minutes in 6000 revs/min, remove supernatant and upper-layer fat, retain precipitation；
D, the precipitation obtained in step c is washed with water one to twice, obtains extract；The method washed with water is：Add Enter 100 DEG C of deionized water 30mL, vibration after mixing, centrifuges 20 minutes in 6000 revs/min, removes supernatant, retains Precipitation；
E, the extract for obtaining step d is freeze-dried, and the temperature of freeze-drying is -47 DEG C, extract after being dried.
Extract example 4：The extracting method of yeast beta-dextran in liquid milk.
A, 25 grams of liquid milks are taken, add 0.016 U of 0.048 U of alkali protease and neutral proteinase, fully vibration mixing When 55 DEG C of heating water baths 3 are small, hydrolyzate is obtained；
B, by hydrolyzate vibration after mixing, centrifuged 20 minutes in 6000 revs/min, remove supernatant, retain precipitation Thing and upper-layer fat；
C, 80 DEG C of deionized water 20mL is added into the obtained sediments of step b and upper-layer fat, vibration is uniformly mixed Afterwards, centrifuged 20 minutes in 6000 revs/min, remove supernatant and upper-layer fat, retain precipitation；
D, the precipitation obtained in step c is washed with water one to twice, obtains extract；The method washed with water is：Add Enter 80 DEG C of deionized water 20mL, vibration after mixing, centrifuges 20 minutes in 6000 revs/min, removes supernatant, and it is heavy to retain Form sediment；
E, the extract for obtaining step d is freeze-dried, and the temperature of freeze-drying is -47 DEG C, extract after being dried.
In said extracted example, alkali protease is purchased from Sigma, article No. P4860-50mL, enzyme activity 2.4U/g；Neutral proteinase Purchased from Sigma, article No. P1236-50mL, enzyme activity 0.8U/g.
Detect example 1：GOPOD methods detect.
First, solution is prepared.
Buffer A（Sodium acetate buffer, pH5,200mM）：11.6mL glacial acetic acid is taken to add in about 900mL water, side edged Stirring.Use sodium hydroxide（4M）PH to 5 is adjusted, is then transferred in 1L volumetric flasks, constant volume.
Buffer B（Sodium acetate buffer, pH3.8,1.2M）：69.6mL glacial acetic acid is taken to add in about 800mL water, side edged Stirring, uses sodium hydroxide（4M）PH to 3.8 is adjusted, is then transferred in 1L volumetric flasks, constant volume.
10 × TES buffer solutions（10×（Carboxymethyl）Methylamine（TRIS）/ EDTA/ salt）：Take 12.12gTRIS, 11.69g chlorine Change sodium, and two water salt of 4.16g sequestrene Na4s（EDTA tetrasodium dihydrate salt）, it is dissolved in about 900ml In pure water, stirred in dissolving.PH is adjusted to 7 with the sodium hydroxide of high concentration HCl or 4M.Solution is moved in 1L volumetric flasks, With water constant volume.
Lywallzyme solution（10U/ μ L are in 1 × TES buffer solutions）：Concentration is 10U/ μ L.Illustrate by factory, take quantitatively molten Wall enzyme is in 10% 10 × TES buffer solutions.Pay attention to：Untapped solution can preserve 1 year under conditions of -15 °.Every time Different with the amount of lyticase, the concentration of lyticase needs to readjust.Wherein, lywallzyme is purchased from Arthrobacter Luteus, Sigma L4025；Other products of same quality can also be used.
1,6- dextranase solution:Lyophilized 1, the 6- dextranases of dissolving are in buffer A, final concentration of 1U/300 μ L. Pay attention to：Solid is not easy fully to dissolve, so this solution needs to be processed into uniform suspension.Solution is at least protected under NMT-15 ° It is 60 days fixed to keep steady.Wherein, 1,6- dextranases are purchased from Pustulanase, Cel136, Prokazyme, or the production of same quality Product.
1,4 beta-glucanase and beta-glucosidase mixed solution：Take circumscribed -1,4 beta-glucanase of 2000U and β-Portugal of 400U Polyglycoside enzyme, with buffer A constant volume to 100mL, passes through at least reverse mixing of ten times.Pay attention to：Prepare and need to be carried out on ice, and And used on the same day.Remaining 1,4 beta-glucanase and beta-glucosidase mixed solution can be frozen in NMT-15 ° again, freeze thawing Only limit once, 2 years terms of validity.Wherein, circumscribed -1,4 beta-glucanase is purchased from E-EXBGL 200U/mL, 200U/bottle, Megazyme, or the product of same quality；Beta-glucosidase is purchased from Megazyme, 200U/bottle, or same quality Product.
Glucose oxidase/peroxidase buffer：45.287g anhydrous potassium dihydrogen phosphate is added in 1 liter of volumetric flask, 30.382g P-hydroxybenzoic acid and 4g sodium azide, the water of careful addition 800mL, is mixed with splash bar and micro- heating, until It is completely dissolved；Solution is poured into a large beaker, pH is adjusted to as 7.4 with the KOH solution of 2M；Solution is refunded again again 1 liter of appearance Measuring bottle water constant volume, overturns mixing at least 6 times.Pay attention to：Buffer storage is in brown bottle in 4 ° of 3 years shelf-lifves.Glucose oxygen Change Bottle#3 (Megazyme) or the GOPOD examination that K-YBGL kits also can be used directly in enzyme/peroxidase buffer The Bottle#1 of agent box（Megazyme）.
Dextran oxidation enzyme/peroxidase reagent（GOPOD solution）：By glucose oxidase/peroxidase of 50mL Buffering, to 1L, adds glucose assays reagent, mixed dissolution with water constant volume.Pay attention to：Reagent is stored in brown bottle, and is marked It is bright, it can be preserved 3 months between 2 DEG C and 8 DEG C in temperature, or preserved 1 year at a temperature of NMT-17 DEG C, preferably avoid in room Warm condition.Glucose assays reagent is the Bottle#2 of the Bottle#4 or GOPOD kits of K-YBGL kits （Megazyme）, or the product of same quality, dosage is with reference to reagent manufacture explanation.
Glucose gradient solution：0 mg/ml, 0.05mg/ml, 0.125 mg/ml , 0.25 mg/ml , 0.5 mg/ The glucose solution of ml；Solvent is water.
2nd, detecting step.
1st, sample digests：
1）, take the drying that said extracted example obtains after extract into centrifuge tube, add 0 DEG C of the KOH solution of 2M 0.4mL, whirlpool mix and are placed on ice bath 20min, obtain translucent homogeneous dispersion soln；Blank control sample is set at the same time（I.e. Extract is empty after drying, remaining simultaneously operating）；
2）, into dispersion soln add 1.6ml buffer Bs, add 600 μ l lywallzyme solution（Lyticase）, slightly Mixing is rocked, acutely can't rock or vibrate, avoids influencing enzyme activity；It is small that lasting 12-18 is placed in 50 DEG C of water-baths after mixing When, be carried out at the same time vortex and shake, sample capping with vaporization prevention, after the completion of be cooled to room temperature, obtain intermediate A；
3）, intermediate A centrifuged 10 minutes under 5000rpm, take 130 μ l supernatants into new test tube, and to new test tube The 2M KOH of 25 μ L of middle addition, mix；300 μ L 1,6- dextranases solution and slight wobble vibration are added, then places 80 Keep 15min in DEG C water-bath, after the completion of be cooled to room temperature, obtain intermediate B；
4）, add 390 μ L 1,4 beta-glucanases and beta-glucosidase mixed solution into intermediate B, 40 DEG C of water-baths insulations 1 After hour, taking-up is cooled to room temperature, and obtains intermediate C；
5）, intermediate C centrifuged into 1.5min, rotating speed 5000rpm, take 50 μ L of supernatant, it is dilute to add 50 μ L deionized waters Release, obtain prepare liquid.
2nd, glucose content measures：
3 mL GOPOD solution, 40 DEG C of water-baths are separately added into prepare liquid obtained above and glucose gradient solution Middle holding 20min；It is finally cooled to room temperature and light absorption value is measured under 510nm, measure uses spectrophotometer, wherein ultraviolet suction Receipts wavelength is 510nm, and detector is Super Emulsion TSM-09, purchased from Sun Chemical Co., Ltd..According to glucose The corresponding light absorption value of gradient solution draws standard curve, and calculates containing for glucose in prepare liquid according to slope of standard curve Amount, specific formula for calculation are as follows：
C=(ABSX-ABSS)/slop；
Wherein：
ABSX：The corresponding light absorption value of prepare liquid；
ABSS：The corresponding light absorption value of blank control sample；
slop：Slope of standard curve；
C:The content of glucose, mg/mL in prepare liquid.
3rd, the content of yeast beta-dextran in liquid milk to be measured is calculated：
X=100 × C/ [（WTs/F1）× (F2/F3)]/2；
Wherein：
X：The content of yeast beta-dextran, mg/100g in liquid milk to be measured；
C:The content of glucose, mg/mL in prepare liquid；
WTs：The quality of liquid milk to be measured, mg；
F1：The volume of intermediate A, 2.6mL；
F2：Step 3）In, the volume of supernatant, 0.130mL are pipetted into new test tube；
F3：The volume of intermediate C, 0.845mL.
Detect example 2：High performance liquid chromatography detects.
First, high-efficient liquid phase chromatogram condition：
Chromatographic column：4 × 50 mm protections of 4 × 250 mm of Dionex CarboPac PA10, band CarboPac PA10 Column；
Mobile phase：A is ultra-pure water, resistivity >=18.2M Ω；B is 0.25 mol/L sodium hydroxide solutions；C is 1 mol/ L sodium acetate solutions；
Detector：Ampere detector；Au working electrodes；Ag/AgCl reference electrodes；30 DEG C of detection cell temperature；Sugar uses Integrated amperometry mode detection, four current potential of waveform selection carbohydrate standard；
Flow velocity：1.0 mL/min；Column temperature：30 ℃；Sample size：25 μL；
Elution gradient see the table below：
Time, min Flow velocity, mL/min OH concentration, mmol/L Sodium acetate concentration, mol/L Gradient curve
0.00 1.0 15 0 Linearly
16.00 1.0 15 0 Linearly
16.10 1.0 100 0.6 Linearly
21.00 1.0 100 0.6 Linearly
21.10 1.0 200 0 Linearly
23.00 1.0 200 0 Linearly
23.10 1.0 15 0 Linearly
30.00 1.0 15 0 Linearly
The sugared waveform parameter of ampere detector detection：
Time (s) Voltage (V) It is integrated
0.000 0.100 off
0.200 0.100 on
0.400 0.100 off
0.410 -2.000 off
0.420 -2.000 off
0.430 0.600 off
0.440 -0.100 off
0.500 -0.100 Off, LastStep on
2nd, detecting step.
1st, sample digests：With the sample enzyme solution of detection example 1, prepare liquid is obtained.
2nd, glucose content detects：
Prepare liquid and glucose gradient solution are detected with high performance liquid chromatography（Glucose gradient solution is shown in detection example 1）. Standard curve is drawn according to the corresponding testing result of glucose gradient solution, and grape in prepare liquid is calculated according to standard curve The content of sugar.
3rd, calculate：
X=100 × C/ [（WTs/F1）× (F2/F3)]/A }；
X：The content of yeast beta-dextran, mg/100g in liquid milk to be measured；
C:The content of glucose, mg/mL in prepare liquid；
WTs：The quality of liquid milk to be measured, mg；
F1：The volume of intermediate A, 2.6mL；
F2：Step 3）In, the volume of supernatant, 0.130mL are pipetted into new test tube；
F3：The volume of intermediate C, 0.845mL；
A：The prepare liquid coefficient of dilution.
Embodiment is set：
Said extracted example and detection example are combined, following examples are set：
Liquid milk sample Extract example Detect example
Embodiment 1 Plain chocolate Extract example 1 Detect example 1
Embodiment 2 Banana milk Extract example 2 Detect example 1
Embodiment 3 Grain milk Extract example 3 Detect example 1
Embodiment 4 Yaourt Extract example 4 Detect example 1
Embodiment 5 Plain chocolate Extract example 1 Detect example 2
Embodiment 6 Banana milk Extract example 2 Detect example 2
Embodiment 7 Grain milk Extract example 3 Detect example 2
Embodiment 8 Yaourt Extract example 4 Detect example 2
Yeast beta-dextran standard items are added into liquid milk sample, mark-on sample are obtained, with the method for above example Mark-on sample is detected and calculates the recovery of standard addition of detection.Testing result is as shown in the table, and unit is " % "：
Embodiment Spiked levels are the recovery of standard addition of 48 mg/100g Spiked levels are the recovery of standard addition of 60 mg/100g Spiked levels are the recovery of standard addition of 78mg/100g
1 97.15 97.39 96.63
2 96.85 95.60 96.83
3 97.46 98.17 96.15
4 96.87 97.58 97.12
5 96.48 96.87 97.84
6 97.33 96.22 98.11
7 97.82 97.72 97.69
8 98.16 96.56 97.36
Yeast beta-dextran standard items are added into liquid milk sample, mark-on sample are obtained, with the method for above example Detection is repeated three times to mark-on sample, and calculate the relative standard deviation of testing result three times, precision is represented with this.Detection As a result as shown in the table, unit is " % "：
Embodiment Spiked levels are the precision of 48 mg/100g Spiked levels are the precision of 60 mg/100g Spiked levels are the precision of 78mg/100g
1 2.36 2.15 2.23
2 3.17 4.03 1.89
3 2.85 3.86 2.35
4 2.67 2.99 2.69
5 4.10 2.98 3.22
6 3.65 2.45 2.69
7 3.73 3.13 2.16
8 3.66 4.23 3.18
Set two comparative examples as follows at the same time：
Comparative example 1：Contrast pre-treating method+present invention and detect example 1；
Comparative example 2：Contrast pre-treating method+present invention and detect example 2；
Wherein, contrast pre-treating method is：Accurately weigh 25.0000g（It is accurate to 0.0002g）Sample is put into one In 150mL centrifuge tubes, 10% potassium ferrocyanide solution of 20% acetic acid zinc solutions of 5mL and 5mL is added, is centrifuged in 7000r/min 10min, removes supernatant, precipitation plus 25mL pure water, and vibration centrifuges 10min after mixing, in 7000r/min, removes supernatant Liquid, is precipitated plus 75% ethanol of 25mL, vibration centrifuge 10min after mixing, in 7000r/min, removes supernatant, precipitation adds again 25mL pure water, vibration centrifuge 10min after mixing, in 7000r/min, remove supernatant.
Recovery of standard addition with the method detection plain chocolate of comparative example 1 and 2 is that unit is " % "：
Embodiment Spiked levels are the recovery of standard addition of 48 mg/100g Spiked levels are the recovery of standard addition of 60 mg/100g Spiked levels are the recovery of standard addition of 78mg/100g
Comparative example 1 93.27 87.61 95.12
Comparative example 2 88.15 92.37 85.16
Precision with the method detection plain chocolate of comparative example 1 and 2 is that unit is " % "：
Embodiment Spiked levels are the precision of 48 mg/100g Spiked levels are the precision of 60 mg/100g Spiked levels are the precision of 78mg/100g
Comparative example 1 5.87 6.14 5.96
Comparative example 2 7.12 6.65 7.56
Herein, " schematic " expression " serving as example, example or explanation ", will should not be described herein as " showing Any embodiment of meaning property " is construed to a kind of preferred or more advantageous technical solution.
Herein, the limitation in " equal ", " identical " etc. and non-critical mathematics and/or geometry meaning, also includes It will be appreciated by those skilled in the art that and production or the error for the permission such as using.Unless otherwise indicated, numerical value model herein The gamut not only included in two endpoint is enclosed, also includes being contained in some subranges therein.
It should be appreciated that although this specification is described according to each embodiment, not each embodiment only includes one A independent technical solution, this narrating mode of specification is only that those skilled in the art will should say for clarity Bright book is as an entirety, and the technical solutions in the various embodiments may also be suitably combined, and forming those skilled in the art can be with The other embodiment of understanding.
Those listed above is a series of to describe illustrating only for possible embodiments of the invention in detail, They simultaneously are not used to limit the scope of the invention, all equivalent embodiments made without departing from skill spirit of the present invention or change More, such as the combination, segmentation or repetition of feature, should all be included in the protection scope of the present invention.

Claims (8)

  1. A kind of 1. extracting method of yeast beta-dextran in liquid milk, it is characterised in that including：
    A, 25 grams of liquid milks are taken, it is mixed to 0.1U and neutral proteinase 0.01 to 0.05U, fully vibration to add alkali protease 0.03 Close when 40 to 60 DEG C of heating water baths 2 to 4 are small, obtain hydrolyzate；
    B, by hydrolyzate vibration after mixing, centrifuged 15 to 20 minutes in 5000 to 6000 revs/min, remove supernatant Liquid, retains sediment and upper-layer fat；
    C, into the obtained sediments of the step b and the upper-layer fat add 60 to 100 DEG C deionized water 15 to 30mL, vibration after mixing, centrifuge 15 to 20 minutes in 5000 to 6000 revs/min, remove supernatant and upper-layer fat, protect Stay precipitation；And
    D, the precipitation obtained in the step c is washed with water one to twice, obtains extract；The side washed with water Method is：Add 60 to 100 DEG C of deionized water 15 to 30mL, vibration and centrifuge 15 after mixing, in 5000 to 6000 revs/min To 20 minutes, remove supernatant, retain precipitation.
  2. 2. the extracting method of yeast beta-dextran in liquid milk as claimed in claim 1, wherein, further include the step d Obtained extract freeze-drying.
  3. 3. the content assaying method of yeast beta-dextran in a kind of liquid milk, it is characterised in that including first passing through claim 1 institute The extracting method of yeast beta-dextran in the liquid milk stated, extracts the yeast beta-dextran in liquid milk to be measured, then measures extraction The content of yeast beta-dextran in product.
  4. 4. the content assaying method of yeast beta-dextran in liquid milk as claimed in claim 3, wherein, including：
    By the extracting method described in claim 1, the yeast beta-dextran in liquid milk to be measured is extracted；
    Product lywallzyme, 1,6- dextranases, circumscribed -1,4 beta-glucanase and the beta-glucosidase that will be obtained after the extraction Hydrolysis, makes yeast beta-dextran all be hydrolyzed into glucose, obtains prepare liquid；And
    The content of glucose in the prepare liquid is measured, and calculates the content of yeast beta-dextran in the liquid milk to be measured.
  5. 5. the content assaying method of yeast beta-dextran in liquid milk as claimed in claim 4, wherein, measure the prepare liquid The method of the content of middle glucose is GOPOD methods.
  6. 6. the content assaying method of yeast beta-dextran in liquid milk as claimed in claim 5, wherein, the GOPOD methods Step includes：3 mL GOPOD solution are added into the prepare liquid, are kept for 20 minutes in 40 DEG C of water-baths；It is finally cooled to room Temperature simultaneously measures absorbance under 510nm.
  7. 7. the content assaying method of yeast beta-dextran in liquid milk as claimed in claim 4, wherein, measure the prepare liquid The method of the content of middle glucose is high performance liquid chromatography.
  8. 8. the content assaying method of yeast beta-dextran in liquid milk as claimed in claim 7, wherein, the high-efficient liquid phase color The condition of spectrometry is：
    Chromatographic column：4 × 50 mm guard columns of 4 × 250 mm of Dionex CarboPac PA10, band CarboPac PA10；
    Mobile phase：A is ultra-pure water, resistivity >=18.2M Ω；B is 0.25 mol/L sodium hydroxide solutions；C is 1 mol/L second Acid sodium solution；
    Detector：Ampere detector；Au working electrodes；Ag/AgCl reference electrodes；30 DEG C of detection cell temperature；Sugar is using integration Ampere mode detection, four current potential of waveform selection carbohydrate standard；
    Flow velocity：1.0 mL/min；Column temperature：30 ℃；Sample size：25 μL；
    Elution gradient see the table below：
    Time, min Flow velocity, mL/min OH concentration, mmol/L Sodium acetate concentration, mol/L Gradient curve 0.00 1.0 15 0 Linearly 16.00 1.0 15 0 Linearly 16.10 1.0 100 0.6 Linearly 21.00 1.0 100 0.6 Linearly 21.10 1.0 200 0 Linearly 23.00 1.0 200 0 Linearly 23.10 1.0 15 0 Linearly 30.00 1.0 15 0 Linearly
    The sugared waveform parameter of ampere detector detection：
    Time, s Voltage, V It is integrated 0.000 0.100 off 0.200 0.100 on 0.400 0.100 off 0.410 -2.000 off 0.420 -2.000 off 0.430 0.600 off 0.440 -0.100 off 0.500 -0.100 Off, LastStep on
    。
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