CN107976503A - A kind of detection method for adding the yeast dextran in albumen powder - Google Patents
A kind of detection method for adding the yeast dextran in albumen powder Download PDFInfo
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- G—PHYSICS
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Abstract
The invention discloses a kind of detection method for adding the yeast dextran in albumen powder, the detection method includes：A) standard curve is drawn；B) extract；C) sample hydrolysis process；D) chromatograph detection sample to be tested；And e) calculate the content for obtaining yeast dextran in sample to be tested.Detection method according to the present invention can exclude the interference of albumen, fat, water-soluble sugar, it is capable of the content of Accurate Determining yeast dextran and the detection method to the content assaying method of yeast dextran by carries out linear, precision test, recovery test, meets GB/T 27,404 2008《Good Laboratory controls specification food Physico-chemical tests》Requirement, it was demonstrated that the detection method is scientific and effective, and the purpose of quality control can be played to the assay of yeast dextran.
Description
Technical field
The present invention relates to analytical chemistry field, more particularly to a kind of detection side for adding the yeast dextran in albumen powder
Method.
Background technology
After big intensity acute exercise, body lymphopoiesis differentiation capability and activity reduce, immunoglobulin content and
Function is also affected, and sportsman and body builder raise the susceptible rate of disease, influence their health or games results.
So the immunosupress after prevention strenuous exercise becomes the important subject of sports medical science, at the same find safely, effectively, it is anti-
Control the sport nutrition material of sports fatigue or method be also athletics sports and nationwide fitness programs there is an urgent need to.
The immune system of the mankind is extremely complex, wherein having thousands of immunocyte to protect human body from thin
The injury of bacterium, pathogen and Other diseases.One of preventing mechanism is exactly reaction of the human body for yeast or fungal infection.This
Some potential harmful microorganisms are the targets of antibody, in antibody binding to these pathogen cells after, will be in guide blood
A kind of soluble protein for being called complement comes in combination with these pathogen cells.Complement can guide neutral grain thin at the same time
Born of the same parents' --- most abundant immunocyte in human body --- are to be attached on pathogen cell.Neutrophil leucocyte is human immune system
The first line of defence, the specific receptor on its surface can identify and be attached to complement combination position.These acceptors also have another
One end, can be with the glucan binding domian that is found in yeast or fungi.The both ends of acceptor must be all by antibody and glucan binding domian
Afterwards, neutrophil leucocyte can be just made to play a role to identify and kill pathogen cell.Therefore, it is exactly that glucan has triggered this kill
Crash system.
There is significant immunoregulation effect from most of polysaccharide of nature extraction, and have and derive from a wealth of sources, the secondary work of poison
With it is low, without carcinogenesis, safe and immune enhancing function is extensive the advantages that, be preferable immunopotentiator.β-1,3-D-
Glucan is a kind of polysaccharide being widely present in microorganism, mushroom and plant, it is composition higher plant, saccharomycete and fungi
One of macromolecular of cell wall structure.Yeast cell wall be tool compared with strong biological activity β -1,3-D- glucans important sources it
One.Yeast cells wall thickness 70nm, accounts for the 20% of dry cell weight.Yeast cell wall is mainly made of 3 kinds of polysaccharide：Mannoproteins
(accounting for 40%), glucan (accounting for 50%) and chitin (about 2%).Yeast dextran includes alkali-insoluble glucan and alkali soluble
Property glucan, wherein alkali-insoluble β -1,3-D- glucan account for 85%.
From tens of thousands of to hundreds of thousands etc., molecular backbone is combined yeast beta-dextran molecular weight with β -1,3 glycosidic bonds, side chain with
β -1,6 glycosidic bonds combine, and this unique triple ultra micro helical structures are the Portugals that immunocompetence is most strong and is most easily absorbed by the body
Glycan form.Numerous studies prove that yeast beta-dextran has enhancing immune function, radioresistance and hypolipemic function.Yeast β-
Glucan has obtained GRAS (Generally Regarded As Safe) certification of U.S. FDA, People's Republic of China's health
Portion have approved yeast beta-dextran as new resource food, this causes yeast beta-dextran also in 2010 by No. 9 bulletin
Have in food service industry and be more widely applied prospect.
At present, country issues there has been no the national examination criteria on yeast beta-dextran and implements.The current Chinese people
Republic light industry standard QB/T4572-2013《Yeast beta-dextran》It is only applicable to using saccharomyces cerevisiae category as raw material, passes through
Breaking-wall cell, acidolysis, the yeast beta-dextran that acid, the technique such as alkali process, separating-purifying, drying and packaging obtain.For addition
As food or the yeast beta-dextran of food ingredient in protein powder etc., standard detecting method there is no.Therefore the present invention
After it with reference to relevant industries standard QB/T 4572-2013 and lot of documents, yeast beta-dextran processing method and color are determined
Spectrum detection method, precision test, recovery test, meet GB/T 27404-2008《Good Laboratory controls specification food
Physico-chemical tests》Requirement.
The content of the invention
In consideration of it, the detection method the present invention provides yeast dextran content in a kind of albumen powder.The detection method can
The interference of the water-solubility impurity such as fat and glucose in albumen powder is excluded, is capable of the content of Accurate Determining yeast dextran；The inspection
It is effective to survey methodological science, the purpose of quality control can be played to the assay of yeast dextran.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：The present invention provides a primary yeast Portugal to gather
Sugared content detection method, is detected using efficient liquid phase chromatographic analysis (HPLC), and the chromatographic condition of HPLC is：Using sugared column or
Nh 2 column, isocratic elution is carried out using purified water or acetonitrile solution as mobile phase, according to HPLC testing results, obtains yeast Portugal
Glycan content.
A) standard curve is drawn：It is accurate to weigh the dissolving of dextrose standard sample water and constant volume, various concentrations are then diluted to,
Measured to obtain glucose standard curve with chromatographic analyzer of liquid phase.
B) extract：Sample to be tested and sodium hydroxide are added to the in the mixed solvent of pure water and absolute ethyl alcohol, after shaking up
Heated in boiling water bath；Centrifuged immediately after, remove supernatant, the pure water mesoscale eddies that precipitation adds 40~50 DEG C is mixed, gone
Except supernatant, test sample is obtained；
C) sample hydrolysis process：Hydrochloric acid solution is added in step b) in obtained precipitation, is vortexed, is uniformly mixed, obtain
Suspension, is then placed in 30 DEG C of water-baths and heats 45 minutes, then put it into 115 DEG C of baking ovens, add by the suspension of homogeneous
When heat 1 is small, cooled down rapidly after taking-up；Hydrolyzate after cooling is adjusted to pH 6~7 with sodium hydroxide solution, and adds water constant volume
And use membrane filtration.
D) under the chromatographic condition identical with standard solution, the sample solution after film excessively and reference substance solution are injected separately into
In chromatograph, the retention time and peak area of each chromatographic peak are recorded.Qualitative using retention time progress, peak area is quantified；
E) content for obtaining yeast dextran in sample to be tested is calculated.
Preferably, the time that boiling water bath heats in step b) is 20~50min, more preferably 30min.
Preferably, the HPLC is more excellent with purified water or acetonitrile solution progress isocratic elution using sugared column or nh 2 column
Selection of land, the nh 2 column specification are that filler particle diameter is 5 μm, long 250mm* diameters 4.6mm.
Preferably, hydrolysising condition is in step c)：Hydrochloric acid is added, 50~100min is hydrolyzed at 100~120 DEG C, is cooled down,
P H values are adjusted to 6.5~7.5 with sodium hydroxide solution；It is further preferred that hydrolysising condition is：Hydrochloric acid is added, at 110 DEG C
90min is hydrolyzed, cooling, pH value is adjusted to 7 with the sodium hydroxide of 10wt%.
Preferably, in hydrolytic process, the mass ratio of yeast dextran and hydrochloric acid is 1:5 to 1:10.
Preferably, the flow rate of mobile phase that HPLC is detected in step d) is 0.5~1.0m L/min, more preferably 1.0m L/
min。
Preferably, the column temperature that HPLC is detected in step d) is 30~40 DEG C, more preferably 35 DEG C.
Preferably, the sample size that HPLC is detected in step d) is 10~30 μ L, more preferably 20 μ L.
Preferably, the run time that HPLC is detected in step d) is 10~20min, more preferably 10min.
Preferably, when using acetonitrile solution as mobile phase, the volume ratio of acetonitrile and water is 75 in acetonitrile solution:25.
Beneficial effect
The present invention can exclude the interference of albumen, fat, water-soluble sugar, be capable of the content of Accurate Determining yeast dextran；This
Invention detection method is accorded with by carrying out linear, precision test, recovery test to the content assaying method of yeast dextran
Close GB/T 27404-2008《Good Laboratory controls specification food Physico-chemical tests》Requirement, it was demonstrated that the detection method science has
Effect, can play the assay of yeast dextran the purpose of quality control.
Brief description of the drawings
Fig. 1 is the representative chromatogram (1.320mg/mL) according to 1 standard solution of embodiment；
Fig. 2 is the representative chromatogram (40mg/g) according to 1 mark-on sample of embodiment；
Fig. 3 is the representative chromatogram according to 1 sample of embodiment；
Fig. 4 is the standard solution linear diagram drawn according to embodiment 2.
Embodiment
Hereinafter, it will be described in detail the present invention.Before doing so, it should be appreciated that in this specification and appended
Claims in the term that uses should not be construed as being limited to general sense and dictionary meanings, and inventor should allowed
On the basis of appropriate definition term is to carry out the principle of best interpretations, according to implication corresponding with the technical elements of the present invention and generally
Thought explains.Therefore, description presented herein is not intended to limitation originally merely for the sake of the preferred embodiment for illustrating purpose
The scope of invention, it will thus be appreciated that without departing from the spirit and scope of the present invention, it can be obtained by it
His equivalents or improved procedure.
Preferably, the preparation method of the test sample of detection method according to the present invention is：Sample is taken into centrifuge tube, is added
Pure water, absolute ethyl alcohol and sodium hydroxide, are heated after shaking up in boiling water bath；Sample after processing is centrifuged immediately, is removed
Supernatant, the pure water mesoscale eddies that precipitation adds 40~50 DEG C mix, remove supernatant, obtain test sample.In the present invention, with nothing
Water-ethanol and sodium hydroxide sample dissolution, make the interfering material such as water-solubility impurity such as fat and glucose in sample carry out effectively
Precipitation separation, can exclusive PCR, the content of Accurate Determining yeast dextran.
PH value is adjusted to 6.5~7.5 with sodium hydroxide solution in the step c) of detection method according to the present invention, is optimized
PH value after the hydrochloric acid additive amount of hydrolyzed yeast glucan and hydrolysis, makes the salinity of sample solution control in suitable scope, both
It ensure that the complete hydrolysis of yeast dextran, in turn ensure that and the consumption of chromatographic column is reduced to minimum, while ensure that salt peak and mesh
There are enough separating degrees at mark peak.
Following embodiments are enumerated only as the example of embodiment of the present invention, do not form any limit to the present invention
System, it will be appreciated by those skilled in the art that the modification in the range of without departing from the essence of the present invention and design each falls within the present invention
Protection domain.Unless stated otherwise, the reagent and instrument used in following embodiments is commercially available product.
Instrument and equipment：Waters2695 highly effective liquid phase chromatographic systems, including quaternary gradient pump, autosampler, column temperature
Case, Waters2410 Composition distributions and Empower 2 operate software (Waters companies, the U.S.)；HPLC NH2 chromatographic columns
(4.6mm × 250mm, 5 μm) (Shiseido companies, Japan)；HWS-26 types electric-heated thermostatic water bath (closes permanent instrument and sets in Shanghai
Standby Co., Ltd)；2 vortex vortex mixers of Vortex Genie (Scientific Industries companies, the U.S.)；
Millipore ultrapure water systems, (Millipore companies of the U.S.)；(Sartorius is public for Sartorius CPA225D electronic balances
Take charge of the U.S.).
Reagent：Yeast beta-dextran (content 70%, Angel Yeast Co., Ltd)；Dextrose standard sample (purity
99.6%, China National Measuring Science Research Inst.)；Methanol, acetonitrile (Merck companies, Germany)；Hydrochloric acid solution (content 37%, Kunshan gold
City reagent Co., Ltd).
With reference to embodiment, the present invention is further explained：
Embodiment 1：The detection of yeast dextran content
1st, liquid phase chromatogram condition：
Table 1-1：Chromatographic condition
2nd, standard solution is prepared
Dextrose standard sample 0.1320g is accurately weighed using a ten thousandth balance, is dissolved in water and is transferred to 100mL capacity
In bottle, scale is diluted with water to, is configured to the standard solution storing solution that concentration is 1.320mg/mL.It is placed in 4 DEG C of preservations.
3rd, the preparation of standard curve
125 μ L of standard reserving solution, 250 μ L, 500 μ L, 750 μ L, 1000 μ L accurately are pipetted, 1mL is diluted to pure water, are prepared
It is respectively the standard solution of 0.165mg/mL, 0.330mg/mL, 0.660mg/mL, 0.990mg/mL, 1.320mg/mL into concentration,
Thus standard working curve is drawn.
4th, sample pre-treatments
Pretreatment before the hydrolysis of 4.1 samples
Sample about 5g (being accurate to 0.01g) accurately is weighed into 200mL centrifuge tubes, adds 45g pure water, the anhydrous second of 50mL
Alcohol, adds 8.0g NaOH, heats 30min after shaking up in boiling water bath after shaking up；Sample after processing is centrifuged immediately,
5000 leave heart 5min, remove supernatant, and precipitation adds 40~50 DEG C of pure water 45mL, is vortexed and mixes, ultrasound 5 minutes, with
10000 revs/min centrifuge 6 minutes, remove supernatant；Precipitation adds 40~50 DEG C of pure water 45mL again, is vortexed and mixes, ultrasound 5
Minute, centrifuged 6 minutes with 10000 revs/min, remove supernatant again, precipitation is spare.
4.2 sample hydrolysis process
Take the hydrochloric acid solution of 25mL, 1mol/L to add in above-mentioned precipitation, be vortexed, be uniformly mixed, obtain the suspension of homogeneous.
Centrifuge tube is put into 30 DEG C of water-baths and heats 45 minutes (every 15 minutes with vortex oscillation 1 time), then puts it into 115 DEG C of baking ovens
In, when heating 1 is small, cooled down rapidly in fume hood after taking-up.Hydrolyzate after cooling is transferred in 100mL volumetric flasks, is used
The sodium hydroxide solution of 10mol/L is adjusted to pH 6~7, and adds water to be settled to scale, is mixed, and is stood.Use 0.45 μm of hole
Membrane filtration is spare.
5th, the measure of sample and reference substance
The measure of 5.1 chromatographies
Under the chromatographic condition identical with standard solution, the sample solution after film excessively and reference substance solution are injected separately into color
In spectrometer, the retention time and peak area of each chromatographic peak are recorded.Qualitative using retention time progress, peak area is quantified.
5.2 results calculate
As a result calculate and use equation below：
In formula：X is beta-dextran content, %；
C：Glucose content in sample solution, mg/mL；
V：The cumulative volume of detected solution, mL；
M：Sample quality unit, g；
0.9：Glucose is converted to glucan coefficient
F：Glucan hydrolysising loss improvement factor, 1.25.
Remarks：The ratio between molecular weight of glucose that glucose obtains after the residue molecular weight and hydrolysis in beta glucan is
0.9, therefore glucose is converted to the coefficient of beta glucan in terms of 0.9.F is destroyed for glucan in sample sour water solution causes result inclined
Low experience penalty coefficient, its value are 1.25.
6th, result of the test
Yeast dextran content detection the results are shown in Table 2 in sample.
Table 2：1 yeast dextran content detection result (results that parallel test is 5 times) of embodiment
Fig. 1 is the representative chromatogram (1.320mg/mL) according to the present embodiment standard solution；Fig. 2 is according to the present embodiment
The representative chromatogram (40mg/g) of mark-on sample；Fig. 3 is the representative chromatogram according to the present embodiment sample.
Embodiment 2：The range of linearity confirms
The test data of standard items is obtained according to 1 detection method of embodiment, is shown in Table 3
Table 3：Standard items test data
Using concentration of standard solution as ordinate, peak area is abscissa, draws standard working curve, as shown in Figure 4.
Linear evaluation：Coefficient R 2 is 0.9994, so with the content of this method indirect determination yeast dextran, dense
Spend good linear to being presented between 1.320mg/mL for 0.165mg/mL, meet GB/T 27404《Good Laboratory control rule
Model food Physico-chemical tests》Requirement【GB/T 27404 requires coefficient R 2 >=0.99】.
Fig. 4 is the standard solution linear diagram drawn according to embodiment 2.
Embodiment 3：Precision and stability test
Replication 5 times under the chromatographic condition of setting with the glucose standards solution of 1.320mg/mL, calculate corresponding
The precision of RSD value expressions, the results are shown in Table 4.Under identical laboratory condition, which repeats 3 within 3 working days
It is secondary, check average value and RSD values, with the stability of day to day precision characterizing method, the results are shown in Table 5.
Table 4：Withinday precision result
Table 5：Day to day precision result
Conclusion：RSD (%) value is can be seen that according to 4 withinday precision result of table and 5 day to day precision result of table to be respectively less than
10%, it was demonstrated that method precision and have good stability, meet Laboratory Request.
Embodiment 4：Accuracy is tested
The accuracy of the method according to the invention is completed by mark-on reclaims experiment, calculates the rate of recovery and RSD values, as a result
It is shown in Table 6.According to standard curve concentration, 3 concentration points of method choice carry out recovery testu, each concentration point parallel determination 5
Part mark-on sample.As a result as shown in table 6-1,6-2,6-3, as can be seen from the table, under three concentration levels, average recovery rate
>70%, meet methodology accuracy requirement.But discovery is tested at the same time, under Individual concentrations level, this method recovery of standard addition
As a result it is relatively relatively low.Therefore, actual sample measure in, if the rate of recovery it is too low (<60%), sample is converted by the rate of recovery
Content in product.
Table 6-1：Rate of recovery experimental result (the horizontal 3.5mg/g of mark-on)
Remarks：The yeast beta-dextran actual content of addition weighs × 70% for addition.
Table 6-2：Rate of recovery experimental result (the horizontal 14mg/g of mark-on)
Remarks：The yeast beta-dextran actual content of addition weighs × 70% for addition.
Table 6-3：Rate of recovery experimental result (the horizontal 28mg/g of mark-on)
Remarks：The yeast beta-dextran actual content of addition weighs × 70% for addition.
Conclusion：Yeast dextran content is detected using detection method according to the present invention, it is its linear, precision test, steady
Qualitative test, accuracy experiment, meet GB/T 27404《Good Laboratory controls specification food Physico-chemical tests》Requirement, card
Bright assay method according to the present invention is scientific and effective, and the purpose of quality control can be played to the assay of yeast dextran.Can
To see, accuracy, precision and the stability of yeast dextran assay method provided by the invention all reach methodology validation requirement,
Can Accurate Determining yeast dextran content.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. the detection method of yeast dextran content in a kind of albumen powder, the detection method uses efficient liquid phase chromatographic analysis
(HPLC) it is detected, the chromatographic condition of HPLC is：Using sugared column or nh 2 column, flowing is used as using purified water or acetonitrile solution
Isocratic elution is mutually carried out, according to HPLC testing results, yeast dextran content is obtained, specifically includes following steps：
A) standard curve is drawn：It is accurate to weigh the dissolving of dextrose standard sample water and constant volume, various concentrations are then diluted to, use liquid
Analysis of hplc instrument measures to obtain glucose standard curve；
B) extract：Sample to be tested and sodium hydroxide are added to the in the mixed solvent of pure water and absolute ethyl alcohol, in boiling water after shaking up
Heated in bath；Centrifuged immediately after, remove supernatant, the pure water mesoscale eddies that precipitation adds 40~50 DEG C mixes, in removal
Clear liquid, obtains test sample；
C) sample hydrolysis process：Hydrochloric acid solution is added in step b) in obtained precipitation, is vortexed, is uniformly mixed, obtain homogeneous
Suspension, then suspension is placed in 30 DEG C of water-baths and is heated 45 minutes, is then put it into 115 DEG C of baking ovens, heating is 1 small
When, cooled down rapidly after taking-up；Hydrolyzate after cooling is adjusted to pH 6~7 with sodium hydroxide solution, and adds water constant volume to be used in combination
Membrane filtration；
D) under the chromatographic condition identical with standard solution, the sample solution after film excessively and reference substance solution are injected separately into chromatography
In instrument, the retention time and peak area of each chromatographic peak are recorded, qualitative using retention time progress, peak area is quantified；
E) content for obtaining yeast dextran in sample to be tested is calculated.
2. detection method according to claim 1, it is characterised in that the time that boiling water bath heats in step b) for 20~
50min, more preferably 30min.
3. detection method according to claim 1, it is characterised in that the nh 2 column specification is that filler particle diameter is 5 μm,
Long 250mm* diameters 4.6mm.
4. detection method according to claim 1, it is characterised in that hydrolysising condition is in step c)：Hydrochloric acid is added,
50~100min is hydrolyzed at 100~120 DEG C, cooling, pH value is adjusted to 6.5~7.5 with sodium hydroxide solution；Further preferably
Ground, hydrolysising condition are：Hydrochloric acid is added, 90min is hydrolyzed at 110 DEG C, is cooled down, pH value is adjusted to 7 with the sodium hydroxide of 10wt%.
5. detection method according to claim 1, it is characterised in that in the hydrolytic process of step c), yeast dextran
Mass ratio with hydrochloric acid is 1:5 to 1:10.
6. detection method according to claim 1, it is characterised in that the flow rate of mobile phase of HPLC detections is in step d)
0.5~1.0m L/min, more preferably 1.0m L/min.
7. detection method according to claim 1, it is characterised in that the column temperature that HPLC is detected in step d) is 30~40
DEG C, more preferably 35 DEG C.
8. detection method according to claim 1, it is characterised in that the sample size that HPLC is detected in step d) is 10~30
μ L, more preferably 20 μ L.
9. detection method according to claim 1, it is characterised in that the run time that HPLC is detected in step d) for 10~
20min, more preferably 10min.
10. detection method according to claim 1, it is characterised in that when using acetonitrile solution as mobile phase, acetonitrile water
The volume ratio of acetonitrile and water is 75 in solution:25.
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