CN104655771A - Method for testing content of galactooligosaccharide in formula milk powder - Google Patents
Method for testing content of galactooligosaccharide in formula milk powder Download PDFInfo
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- CN104655771A CN104655771A CN201410415976.5A CN201410415976A CN104655771A CN 104655771 A CN104655771 A CN 104655771A CN 201410415976 A CN201410415976 A CN 201410415976A CN 104655771 A CN104655771 A CN 104655771A
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- galactose
- galactooligosaccharide
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- lactose
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- 235000021255 galacto-oligosaccharides Nutrition 0.000 title claims abstract description 57
- 150000003271 galactooligosaccharides Chemical class 0.000 title claims abstract description 57
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- 239000000843 powder Substances 0.000 title abstract description 11
- 238000012360 testing method Methods 0.000 title abstract description 6
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- 239000008101 lactose Substances 0.000 claims abstract description 36
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- 239000007788 liquid Substances 0.000 claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 19
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- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims abstract description 14
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- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 claims description 14
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- XCOBLONWWXQEBS-KPKJPENVSA-N N,O-bis(trimethylsilyl)trifluoroacetamide Chemical compound C[Si](C)(C)O\C(C(F)(F)F)=N\[Si](C)(C)C XCOBLONWWXQEBS-KPKJPENVSA-N 0.000 abstract description 4
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- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 4
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- FFUAGWLWBBFQJT-UHFFFAOYSA-N hexamethyldisilazane Chemical compound C[Si](C)(C)N[Si](C)(C)C FFUAGWLWBBFQJT-UHFFFAOYSA-N 0.000 description 2
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- 125000002519 galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 description 1
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- Saccharide Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for testing content of galactooligosaccharide, and in particular relates to a method for testing content of galactooligosaccharide in formula milk powder by means of gas chromatography-mass spectrum. The method comprises the following steps: extracting galactooligosaccharide and lactose from samples to be detected by using hot phosphate buffer liquid; hydrolyzing and centrifuging galactooligosaccharide in extracting liquid by using beta-galactosidase to obtain liquid supernatant; enabling the liquid supernatant to pass through a microfiltration membrane; carrying out nitrogen blowing or performing low-temperature vacuum drying; deriving by using BSTFA and making up to the volume by using acetone. The content of dissociative galactose and lactose in extracting raw liquid and the total quantity of galactooligosaccharide obtained by enzymolysis on decomposition liquid and galactose released by lactose are tested by utilizing a gas phase-mass spectrum combined method, and the content of the galactooligosaccharide in samples is calculated by using the content of galactose content obtained by performing enzymolysis on decomposition liquid to substract the content of galactose obtained by extracting the raw liquid and the content of galactose produced by lactase enzymolysis. The content of galactooligosaccharide in the milk powder is detected by using the method, and the method has the advantages of extremely-low detection and high sensitivity.
Description
This method relates to the detection method of Determination of galactooligosacchariin in Gas Chromatography-Mass Spectrometry formula milk, belongs to dairy products detection field.
Background technology
Galactooligosaccharide, English name Galactooligosaccharide, be called for short GOS, it is a kind of functional oligose with natural attribute, its molecular structure is generally connect 1-7 galactosyl on galactose (Gal) or glucose (Glc) molecule, i.e. Gal-(Gal) n-Glc/Gal (n is 0-6), molecular weight is about 300-2000.Under the chemical structural formula of galactooligosaccharide is shown in:
The chemical structural formula of galactooligosaccharide
Galactooligosaccharide content in lacto is more, and the foundation of the species population in infants relies on the GOS composition in breast milk to a great extent.GOS can not be digested and assimilated by human body alimentary canal, directly can enter large intestine, can utilize by 8 large beneficial bacteriums in human body intestines, the growth and breeding of human body intestinal toxic bacterium can be suppressed, have stronger acid resistance, thermotolerance.Research is thought, galactooligosaccharide have low-yield, promote the propagation of intestinal bifidobacteria, improve the absorption of mineral matter element and prevent sclerotin from reducing, improving lipid-metabolism, prevention, treatment constipation, generate nutriment, improve nutrition condition, improve immunity, strengthen antitumor and anti-ageing trophic function of waiting for a long time.Galactooligosaccharide has extensively been used as substitute, raw-food material, the functional food ingredient of sweetener, sugar in Japan.Galactooligosaccharide is also called as the prebiotics of breast milk, one of formula becoming infant food.
China does not still have in food now, especially the bioassay standard method of galactooligosaccharide in formula milk.It is food additives new varieties that the Ministry of Public Health of former China (existing national health State Family Planning Commission) ratifies galactooligosaccharide in No. 12 bulletin in 2007, and usable range is infant formula, larger baby and baby formula.The total amount of such material in dispensed food for baby, larger baby and baby formula is no more than 64.5g/kg.It is that new resource food is processed for food production that the former Ministry of Public Health proposes approval galactooligosaccharide according to the regulation of " Food Hygiene Law of the People's Republic of China " and " new resource food management method " in No. 20 bulletin in 2008, the scope of application is infant food, dairy products, beverage, bakery product, candy, and amount is not more than 15g/d.
That commonly uses both at home and abroad at present has Slegte J method [Slegte J.Determination of trans-Galactooligosaccharides in selected food products by Ion-Exchange Chromatography:collaborative study, J AOAC intern, 2002, 2 (85): 417-423], the method is adopted by U.S. AOAC, namely AOAC2001.02 measures transgalactooligosac,harides ion-exchange chromatography [the AOAC Offiical Method45.4.12-2001.02 in certain food, Determination of trans-Galactooligosaccharides (TGOS) in selected food product, Ion-Exchange Chromatography.JAOAC intern, 2002], other countries not yet issue standard method to galactooligosaccharide.The Method And Principle of AOAC2001.02 from testing sample, extracts GOS and lactose, with beta galactosidase by the GOS in extract and lactose hydrolysis with the phosphate buffer of heat; Pulsed amperometry high performance anion exchange chromatography (HPAEC-PAD) is used to detect galactose free in stoste and lactose respectively by extracting stoste Sum decomposition liquid, the galactose total amount that in Sum decomposition liquid, GOS discharges, can calculate the amount of GOS from the concentration of lactose and galactose.
The mensuration of galactooligosaccharide " in the food " GB exposure draft [state defends and does No. (2014) 385, food letter] that on May 7th, 2014, general office of national health State Family Planning Commission issued, define the method for galactooligosaccharide in food by ion chromatography in method, be applicable to the mensuration of galactooligosaccharide in the food such as infant food, dairy products, bakery product, candy, beverage, vinegar, syrup, Icing Sugar.But method is not suitable for the product that in product, lactose content and Determination of galactooligosacchariin ratio are greater than 6, quantitatively being limited to of method: milk powder is 50.0g/kg, infant food, bakery product are 3.50g/kg, and liquid milk, candy, beverage, vinegar are 10.0g/kg, and syrup, Icing Sugar are 20.0g/kg.The method galactooligosaccharide quantitative limit is too high, is not suitable for some and contains the lower sample of galactooligosaccharide.
Because galactooligosaccharide belongs to compound sugar, and the separation detection technique of compound sugar is difficult point and the emphasis of current research always.The detection of current galactooligosaccharide mainly contain thin-layered chromatography (TLC), high performance liquid chromatography (HPLC), GC-MS (GC-MS), high-efficient ion chromatogram method (HAPEC), high performance liquid chromatography tandem ion-trap mass spectroscopy, liquid chromatography tandem electrospray ionization mass spectrometry (LC-ESI-MS/MS) to galactooligosaccharide carry out separation andpreconcentration [Zhang Zhi state. galactooligosaccharide detection method progress. food research and development, 2013,34 (8): 115-120].Monose is separated with compound sugar HPLC by Hirotaka Kakita etc., then carry out the derivative rear fluorescence detector of fluorescein to detect, achieve good effect [Hirotaka Kakita, et al.Simultaneous analysis of monosaccharides and oligosaccharides by high performance liquid chromatography with postcolumn fluorescence derivatization.Chromatogr A, 2002,961 (1): 77-8)].
High effective liquid chromatography for measuring GOS is mainly separated with on sugared post, nh 2 column or gel column, adopt differential refraction detector (RID) or detect by evaporative light-scattering detector (ELSD), ELSD detects limit for height 1-2 the order of magnitude than differential refraction detector.Zhao Biner adopts beta galactosidase galactooligosaccharide to carry out enzymolysis, nh 2 column is separated, by high performance liquid chromatography-differential refraction detector measure lactose and galactose before and after infant formula enzymolysis of poor quality come galactooligosaccharide total content in calculation sample.Best Pretreatment is: weigh sample quality and be less than 10g, during extraction, phosphate buffer solution consumption is 40mL, enzyme concentration is 1.0mL, enzymolysis time is 30min, hydrolysis temperature is 60 DEG C, recovery of standard addition the highest by 82.4% (Zhao Biner. the research of galactooligosaccharide method in high-performance liquid chromatogram determination infant formula, 2013,05:317).Zhang Zhi states etc. adopt galactooligosaccharide [the Zhang Zhi state in HPLC-ELSD method detection liquid milk, the .HPLC-ELSD methods such as Wang Shuo detect the galactooligosaccharide in liquid milk. food and machinery, 2011,27 (3): 68-70,75], this article discloses the method for galactooligosaccharide in a kind of liquid milk.Sample, by beta galactosidase, glucose oxidase-peroxidase process, adopts the galactose content in HPLC-ELSD detection hydrating solution, calculates the content of galactooligosaccharide in sample.Detecting of the method is limited to 0.10mg/mL, and the recovery is 87.5% ~ 108.3%.Li Jing's virtue is separated with Peng Meichun liquid chromatography nh 2 column, content [Li Jing's virtue of galactooligosaccharide in differential pulse polarograpll syrup and dairy products, Peng Meichun. the content of high effective liquid chromatography for measuring galactooligosaccharide. food science and technology, 2012,37 (7): 279:283.].Also the useful chromatography of ions directly measure syrup gather lactose, galactose, glucose and galactooligosaccharide report (Xu Li etc. the assay method of Determination of galactooligosacchariin. food and fermentation industries, 2011,2:179-181).
To the mensuration of monose in polysaccharide, carry out gas chromatographic analysis, must first convert it into volatile, to the more stable derivant of heat, common derivatization method is that silanization is (as hexamethyldisilazane+trimethyl chlorosilane; Hexamethyl four silicon amine alkane+trimethyl chlorosilane etc.), monose (reduces by acetylation (acetic anhydride) or alcohol sugar ethyl ester derivatization, acetylation generates alcohol sugar ethyl ester), then [white sister-in-law Si is separated with capillary chromatographic column, Zhang Jing. the research of gas chromatographic analysis polysaccharide derivatization method with compare. food industry science and technology, 2,322-324; He Yonghui etc., acetic anhydride Derivative GC method measures the monose composition of wheat SNSP, spectrographic laboratory, 2009,, but in document, have no the gas chromatography-mass spectrography analyzing galactooligosaccharide in formula milk 26 (04): 1039-1042].
To sum up show, the mode that generally galactooligosaccharide can only be made to be converted into galactose and glucose by enzyme hydrolysis now calculates the content of galactooligosaccharide, namely deduct the galactose content of free galactose content and Lactis Anhydrous hydrolysis generation by the total amount of galactose after hydrolysis, thus calculate the content of galactooligosaccharide in sample.Although the instrument detection technique of galactooligosaccharide has liquid chromatography, chromatography of ions etc., but be often added with to the formula milk of the prebiotics factor galactooligosaccharide recent years, because its matrix is complicated, if Impurity removal is bad in sample pretreatment process, the measurement result of interference Instrument, in addition liquid chromatography and chromatography of ions sensitivity not high, can not accurate quantitative analysis to the lower galactose of content.Simultaneously because need to carry out Determination of galactooligosacchariin in calculation sample by the galactose increment in working sample, lactose free in sample, galactose content are the key factors affecting measurement result accuracy, because lactose content in milk powder is too high, relatively high by the galactose increment obtained after hydrolysis, error at measurment is larger.Survey the galactooligosaccharide in milk powder by AOAC method, method is consuming time, loaded down with trivial details, and cost is higher, and precision is lower, disturbs larger.And in an experiment, we also tested HPLC method or HPAEC method detects, but interference is comparatively large, and especially glucose and galactose are difficult to separately.
How exactly quantitative lactose, galactose content, become the bottleneck that in dairy products, Determination of galactooligosacchariin is analyzed, and needed to reduce detection limit and quantitative lower bound energetically, this is also the problem that the present invention mainly solves.At present both at home and abroad also not for the assay method of the gas chromatography-mass spectrum of galactooligosaccharide in formula milk.
Summary of the invention
Object of the present invention is exactly the deficiency overcoming existing analytical approach, sets up a kind of fast and accurately to the assay method of galactooligosaccharide in formula milk;
The present invention proposes the gas chromatography-mass spectrum detection method of galactooligosaccharide in a kind of formula milk, the method comprises the following steps:
(1) extract: weigh 1.0g (being accurate to 0.01g) homogeneous sample and join in 50mL graduated plastic volumetric flask, with 40mL phosphate buffer (0.2M phosphate buffer: 22.0g potassium dihydrogen phosphate+6.0g dipotassium hydrogen phosphate+1.0L secondary water) constant volume, 80 DEG C ± 2 DEG C are extracted lasting jolting in water-bath and extract 30min, 100mL is settled to by phosphate buffered solution after being cooled to room temperature, pipette the extraction solution of 5.0mL sample in 50mL volumetric flask, 50mL is settled to by phosphate buffered solution, for subsequent use.
(2) enzymolysis: beta galactosidase solution preparation, take the beta galactosidase that activity is about 500U and be dissolved in 10mL phosphate buffered solution, be finally diluted to the final active beta galactosidase solution for 50U/mL of preparation, the used time now joins.Removing step (1) 1.0mL extract is in 10mL volumetric flask, add 0.5mL beta galactosidase solution, mixing, sustained oscillation 60min in 60 DEG C ± 2 DEG C water-baths, is settled to 10mL with 20% acetonitrile after being cooled to room temperature, shake up, the centrifugal 5min of 10000r/min, upper strata aqueous phase Filter paper filtering, with 0.22 μm of aqueous phase membrane filtration, wait to derive, measure and the concentration (C of galactose after calculating enzymolysis
1).
(3) before enzymolysis: pipette extract 1.0mL in 10mL graduated plastic centrifuge tube simultaneously, add 0.5mL phosphate buffered solution, mixing, continues jolting 60min, is settled to 10mL after being cooled to room temperature with 20% acetonitrile in 60 DEG C ± 2 DEG C water-baths, shake up, the centrifugal 10min of 10000r/min, upper strata aqueous phase Filter paper filtering, then uses 0.22 μm of aqueous phase membrane filtration, wait to derive, measure and calculate the concentration (C extracting free lactose in stoste
2) and the concentration (C of free galactose
3).
(4) derivative: to get final sample liquid in step (2) and (3) respectively and to dry up or after the drying of low temperature (50-60 DEG C) vacuum dryer through nitrogen, add 400 μ LN, dinethylformamide (DMF) and 200 μ L derivative reagent (N, the mixed liquor (99: 1 of two (TMS) trifluoroacetamide (BSTFA) of O-and trimethyl chlorosilane (TMCS), v/v) concussion mixing, after derivative reagent adds, ensure hermetically drying, derivative 30min at 75 DEG C ± 2 DEG C, after cooling, 1mL is settled to acetone, cross 0.22 μm of organic film.
(5) measure: the solution after constant volume enters gas chromatograph-mass spectrometer (GCMS), Selective ion mode scanning (SIM) detects, quantified by external standard method.
(6) as preferred, gas chromatography-mass spectrum condition is as follows:
Adopt gas chromatograph-mass spectrometer (GCMS), join electron bombardment ionization source (EI);
Chromatographic column: DB-5MS capillary gas chromatography mass spectrum post (30m × 0.25mm × 0.25 μm) or quite post;
Chromatographic condition is: injector temperature 280 DEG C, transmission line temperature 150 DEG C, helium flow velocity is 1.0mL/min, sample size 1 μ L, split ratio=20: 1, solvent delay 5min, heating schedule: 150 DEG C, be warming up to 230 DEG C with 4 DEG C/min, be warming up to 280 DEG C (keeping 15min) with 20 DEG C/min;
Detection mode: SIM
As preferably, the retention time of lactose, galactose, qualitative ion, quota ion are as follows:
Note: galactose-1, galactose-2, galactose-3 is the isomers of galactose derivative, and lactose-1, lactose-2 are the isomers of lactose derivatives.
(7) computing formula:
In sample, Determination of galactooligosacchariin X presses formula (a) calculating, and numerical value represents with mg/100g.
In formula:
X---Determination of galactooligosacchariin in sample, unit is the every hectogram of milligram (mg/100g);
1.35---the conversion factor factor;
Note: 1.35 is the experience k value of galactooligosaccharide raw material, and different products may have difference, the k value of the galactooligosaccharide raw material actual measurement added by reality is as calculated value.
G
t---galactose content final in enzymolysis decomposed solution, unit is the every hectogram of milligram (mg/100g);
L
b---the lactose content in initial extraction liquid, unit is the every hectogram of milligram (mg/100g);
1.9---lactose is converted into the experience reduction coefficient of galactose;
G
b---the galactose content in initial extraction liquid, unit is the every hectogram of milligram (mg/100g).
G in formula (a)
t, L
b, G
bcalculate according to formula (b)-(e):
In above-mentioned formula:
G
t---galactose content final in enzymolysis decomposed solution, unit is the every hectogram of milligram (mg/100g);
C
1---the concentration of galactose final in enzymolysis decomposed solution, unit is micrograms per millilitre (μ g/mL);
L
b---extract the lactose content in stoste, unit is the every hectogram of milligram (mg/100g);
C
2---extract the concentration of the lactose in stoste, unit is micrograms per millilitre (μ g/mL);
G
b---the galactose content in extract, unit is the every hectogram of milligram (mg/100g);
C
3---extract the concentration of the galactose in stoste, unit is micrograms per millilitre (μ g/mL);
M---sample sample weighting amount, unit is gram (g);
V---sample handling processes dilution volume, unit is milliliter (mL);
10---unit conversion factor.
Result of calculation represents with the arithmetic mean of twice that obtains under repeated condition independent measurement result, retains 3 position effective digitals.
The absolute difference of twice that obtains under repeated condition independent measurement result must not exceed 10% of arithmetic mean.
(8) detection limit and quantitative limit: detection limit and the quantitative limit of distinguishing computing method according to signal to noise ratio (S/N ratio)=3 and signal to noise ratio (S/N ratio)=10, the method detection limit and quantitative limit are respectively 7mg/100g and 20mg/100g, or 70mg/kg and 200mg/kg.
Beneficial effect of the present invention:
(I) the present invention adopts galactooligosaccharide, galactose, the lactose in phosphate buffer extraction milk powder product at a certain temperature, and extraction effect is good, simple to operate;
(II) through method optimization, sample size used is few, and beta galactosidase amount ratio AOAC enzymatic isolation method reduces by more than 10 times.
(III) adopt GC-MS method to carry out galactose and lactose detects, the range of linearity is wide, have interference less, detection limit is low, degree of accuracy is high.
(IV) for the formula milk of common interpolation galactooligosaccharide on the market, before enzymolysis, extract and enzymolysis decomposed solution can require no dilution, simplify analytical procedure.
Accompanying drawing illustrates:
Fig. 1 is the chromatogram of 50 μ g/mL standard solutions, and wherein galactose-1, galactose-2, galactose-3 are the derivant isomers of galactose, and lactose-1, lactose-2 is the derivant isomers of lactose.
Embodiment:
Below by embodiment, technical scheme of the present invention is further described.
The Gas Chromatography-Mass Spectrometry of Determination of galactooligosacchariin in embodiment 1 baby formula milk powder
(1) extract: take sample 1.0g (being accurate to 0.0001g) in 50mL graduated plastic centrifuge tube, add 40mL phosphate buffered solution (0.2M phosphate buffer: 22.0g potassium dihydrogen phosphate+6.0g dipotassium hydrogen phosphate+1.0L secondary water) to dissolve, vortex mixes.Sustained oscillation 30min in 80 DEG C ± 2 DEG C water-baths, is settled to 100mL by phosphate buffered solution after being cooled to room temperature.The extraction solution pipetting 5.0mL sample, in 50mL volumetric flask, is settled to 50mL by phosphate buffered solution, for subsequent use.
(2) enzymolysis: simultaneously pipette extract 1.0mL in 10mL volumetric flask, add 0.5mL beta galactosidase solution (50U/mL), mixing, sustained oscillation 60min in 50 DEG C ± 2 water-baths, is settled to 10mL with 20% acetonitrile after being cooled to room temperature, shakes up, the centrifugal 10min of 10000r/min, upper strata aqueous phase Filter paper filtering, with 0.22 μm of aqueous phase membrane filtration, waits to derive.
(3) before enzymolysis: pipette extract 1.0mL in 10mL volumetric flask, add 0.5mL phosphate buffered solution, mixing, sustained oscillation 60min in 60 DEG C ± 2 DEG C water-baths, is settled to 10mL with 20% acetonitrile after being cooled to room temperature, shakes up, the centrifugal 10min of 10000r/min, upper strata aqueous phase Filter paper filtering, with 0.22 μm of aqueous phase membrane filtration, waits to derive.
(4) derivative: the final sample liquid 1mL pipetting (2) and (3) respectively, nitrogen dries up, add 400 μ L DMF respectively, 200 μ L derivative reagent BSTFA and: the mixed liquor (99: 1, v/v) of TMCS, 75 DEG C ± 2 DEG C derivative 30min, after cooling, be settled to 1mL with acetone, cross 0.22 μm of organic film, detect with gas-mass spectrometer.
(5) measure: the solution after constant volume enters gas chromatograph-mass spectrometer (GCMS), Selective ion mode Scanning Detction, quantified by external standard method;
(6) Agilent 7890A-5975C gas chromatograph-mass spectrometer (GCMS); Gas chromatography-mass spectrum condition: chromatographic column: DB-5MS capillary column, 30m × 0.25mm × 0.25 μm); Injection port: 280 DEG C; Transmission line temperature: 280 DEG C; Helium flow velocity is 1.0mL/min; Input mode: shunting, split ratio 20: 1; Sample size 1 μ L; Solvent delay 5min; Column temperature: 150 DEG C; Heating schedule: be warming up to 230 DEG C with 4 DEG C/min from 150 DEG C, is warming up to 280 DEG C (keeping 15min) with 20 DEG C/min; Carrier gas: helium; Flow velocity: 1.0mL/min; Ionization mode: EI; Ionizing energy: 70eV; Mensuration mode: SIM; Solvent delay: 5min; Ion source temperature: 230 DEG C.
The retention time of lactose, galactose derivative, qualitative ion, quota ion are in table 1.
The retention time of table 1 lactose, galactose derivative, qualitative ion, quota ion are as follows:
Note: galactose-1, galactose-2, galactose-3 is the isomers of galactose derivative, and lactose-1, lactose-2 are the isomers of lactose derivatives.
(7) standard items is derivative: be 2 μ g/mL, 5 μ g/mL, 50 μ g/mL, 100 μ g/mL, the galactose of 200 μ g/mL and lactose mixed standard solution with chromatogram pure water compound concentration, get in above-mentioned each solution 0.1mL and test tube respectively with transfer pipet, then operate according to the requirement of upper (5) step, according to the standard concentration added (X) and peak area (Y) drawing standard curve; Galactose and lactose concentration of standard solution and corresponding peak area thereof respectively in table 2 and table 3, equation of linear regression and R
2be respectively Y=46364X-50884, R
2=0.9975, Y=30626X+57649, R
2=0.9997.
Table 2 galactose concentration of standard solution and corresponding peak area thereof
Table 3 lactose concentration of standard solution and corresponding peak area thereof
(7) mensuration of recovery of standard addition
This powdered milk sample galactooligosaccharide does not detect.In negative formula milk sample, add the mixed standard solution of galactose and lactose respectively by the concentration of 10mg/g and 20mg/g, each Pitch-based sphere does six Duplicate Samples respectively; By step (1)-(6) operation, method recovery of standard addition is at 94.3-102%, RSD≤10%.
With ten times of snr computation the method quantitative limit.The method is quantitatively limited to 20mg/100g.
The Gas Chromatography-Mass Spectrometry of galactooligosaccharide in embodiment 2 infant formula goat milk powder 2 sections
(1) sample extracts: take infant formula goat milk powder 2 sections of 1.0003g and 1.0010g respectively, and constant volume adds the dissolving of 40mL phosphate buffered solution in 100mL volumetric flask respectively, and vortex mixes.Sustained oscillation 30min in 80 DEG C ± 2 DEG C water-baths, is settled to 100mL by phosphate buffered solution after being cooled to room temperature.The extraction solution pipetting 5.0mL sample, in 50mL volumetric flask, is settled to 50mL by phosphate buffered solution, for subsequent use.
(3) enzymolysis: pipette extract 1.0mL in 10mL volumetric flask, add 0.5mL beta galactosidase solution (50U/mL), mixing, sustained oscillation 60min in 50 DEG C of water-baths, is settled to 10mL with 20% acetonitrile after being cooled to room temperature, shakes up, the centrifugal 10min of 10000r/min, upper strata aqueous phase Filter paper filtering, with 0.22 μm of aqueous phase membrane filtration, waits to derive.
(4) before enzymolysis: pipette extract 1.0mL in 10mL volumetric flask simultaneously, add 0.5mL phosphate buffered solution, mixing, sustained oscillation 60min in 60 DEG C ± 5 DEG C water-baths, is settled to 10mL with 20% acetonitrile after being cooled to room temperature, shakes up, the centrifugal 10min of 10000r/min, upper strata aqueous phase Filter paper filtering, with 0.22 μm of aqueous phase membrane filtration, waits to derive.
(5) analyte derivative: pipette final sample liquid 1mL, nitrogen dries up, add 400 μ L DMF respectively, 200 μ L derivative reagent BSTFA and: the mixed liquor (99: 1, v/v) of TMCS, 75 DEG C ± 2 DEG C derivative 30min, after cooling, be settled to 1mL with acetone, cross 0.22 μm of organic film, detect with gas-mass spectrometer.
(6) gas chromatography-mass spectrum condition: Agilent7890A-5975C gas chromatograph-mass spectrometer (GCMS); Chromatographic column: DB-5MS capillary column, 30m × 0.25mm × 0.25 μm); Injection port: 280 DEG C; Transmission line temperature: 280 DEG C; Helium flow velocity is 1.0mL/min; Input mode: shunting, split ratio 20: 1; Sample size 1 μ L; Solvent delay 5min; Heating schedule: 150 DEG C, is warming up to 230 DEG C with 4 DEG C/min, is warming up to 280 DEG C (keeping 15min) with 20 DEG C/min; Carrier gas: helium; Flow velocity: 1.0mL/min; Ionization mode: EI; Ionizing energy: 70eV; Mensuration mode: SIM; Solvent delay: 5min; Ion source temperature: 230 DEG C; And qualitative according to retention time, the qualitative ion of isomers of lactose and galactose derivative is respectively m/z191 and m/z217, take m/z204 as quota ion.
(7) testing result
Larger infant formula goat milk powder 2 sections: Determination of galactooligosacchariin is respectively 620.4 and 619.1mg/100g, mean value 620mg/100g.
Above-described embodiment is used for illustrative purposes only, and is not the restriction to patent of the present invention; It should be pointed out that for those of ordinary skill in the art, when not departing from concept of the present invention, can also make various change and modification, these all belong to protection scope of the present invention; Therefore, all equalizations done with the claims in the present invention scope change and modify, and all should belong to the coverage of the claims in the present invention.
Claims (2)
1. measure a detection method for Determination of galactooligosacchariin in formula milk, it is characterized in that the method comprises the following steps:
(1) extract: take homogeneous sample 1.0g (being accurate to 0.01g) in 50mL graduated plastic centrifuge tube, add 40mL phosphate buffer (0.2M phosphate buffer: 22.0g potassium dihydrogen phosphate+6.0g dipotassium hydrogen phosphate+1.0L secondary water) to dissolve, vortex mixes, in 80 DEG C ± 2 DEG C water-baths, continue jolting extract 30min, 100mL is settled to by phosphate buffered solution after being cooled to room temperature, pipette the extraction solution of 5.0mL sample in 50mL volumetric flask, 50mL is settled to by phosphate buffered solution, for subsequent use.
(2) enzymolysis: pipette extract 1.0mL in 10mL graduated plastic centrifuge tube, add 0.5mL beta galactosidase solution (deriving from aspergillus oryzae, 50U/mL), mixing, jolting 60min is continued in 60 DEG C ± 2 DEG C water-baths, be settled to 10mL with 20% acetonitrile after being cooled to room temperature, shake up, the centrifugal 10min of 10000r/min, upper strata aqueous phase Filter paper filtering, with 0.22 μm of aqueous phase membrane filtration, wait to derive, measure and the concentration (C of galactose after calculating enzymolysis
1).
(3) before enzymolysis: pipette extract 1.0mL in 10mL graduated plastic centrifuge tube simultaneously, add 0.5mL phosphate buffered solution, mixing, continues jolting 60min, is settled to 10mL after being cooled to room temperature with 20% acetonitrile in 60 DEG C ± 2 DEG C water-baths, shake up, the centrifugal 10min of 10000r/min, upper strata aqueous phase Filter paper filtering, then uses 0.22 μm of aqueous phase membrane filtration, wait to derive, measure and calculate the concentration (C extracting free lactose in stoste
2) and the concentration (C of free galactose
3).
(4) derivative: the final sample liquid 1mL pipetting (2) and (3) respectively, nitrogen dries up or after low temperature (50 ~ 60 DEG C) vacuum drying, add 400 μ L N respectively, dinethylformamide (DMF) and 200 μ L derivative reagent (N, (ratio is 99: 1 to the mixed liquor of two (TMS) trifluoroacetamide of O-and trimethyl chlorosilane, v/v), ensure seal and shake mixing, 75 DEG C ± 2 DEG C derivative 30min, after cooling, be settled to 1mL with acetone, cross 0.22 μm of organic film.
(5) measure: the solution after constant volume enters gas chromatograph-mass spectrometer (GCMS), Selective ion mode Scanning Detction (SIM), quantified by external standard method;
(6) adopt gas chromatograph-mass spectrometer (GCMS), join electron bombardment ionization source (EI); As preferably, gas chromatography-mass spectrum condition: chromatographic column: DB-5MS capillary column (30m × 0.25mm × 0.25 μm) or quite post; Injection port: 280 DEG C; Transmission line temperature: 280 DEG C; Helium flow velocity is 1.0mL/min; Input mode: shunting, split ratio 20: 1; Sample size 1 μ L; Column temperature: 150 DEG C; Heating schedule: be warming up to 230 DEG C with 4 DEG C/min from 150 DEG C, is warming up to 280 DEG C (keeping 15min) with 20 DEG C/min; Carrier gas: helium; Flow velocity: 1.0mL/min (constant current); Ionization mode: EI; Ionizing energy: 70eV; Mensuration mode: SIM; Solvent delay: 5min; Ion source temperature: 230 DEG C; And qualitative according to retention time, the qualitative ion of the derivant of galactose and lactose is all m/z 191 and m/z 217, with m/z 204 for quota ion.
(7) computing formula:
In sample, Determination of galactooligosacchariin X presses formula (a) calculating, and numerical value represents with mg/100g.
In formula:
X---Determination of galactooligosacchariin in sample, unit is the every hectogram of milligram (mg/100g);
1.35---the conversion factor factor;
Note: 1.35 is the experience k value of galactooligosaccharide raw material, and different products may have difference, the k value of the galactooligosaccharide raw material actual measurement added by reality is as calculated value.
G
t---galactose content final in enzymolysis decomposed solution, unit is the every hectogram of milligram (mg/100g);
L
b---the lactose content in extract stoste, unit is the every hectogram of milligram (mg/100g);
1.9---lactose is converted into the experience reduction coefficient of galactose;
G
b---extract the galactose content in stoste, unit is the every hectogram of milligram (mg/100g).
G in formula (a)
t, L
b, G
bcalculate according to formula (b)-(e):
In above-mentioned formula:
G
t---galactose content final in enzymolysis decomposed solution, unit is the every hectogram of milligram (mg/100g);
C
l---galactose concentration final in enzymolysis decomposed solution, unit is micrograms per millilitre (μ g/mL);
L
b---extract the lactose content in stoste, unit is the every hectogram of milligram (mg/100g);
C
2---extract the concentration of the lactose in stoste, unit is micrograms per millilitre (μ g/mL);
G
b---extract the galactose content in stoste, unit is the every hectogram of milligram (mg/100g);
C
3---extract the galactose concentration in stoste, unit is micrograms per millilitre (μ g/mL);
M---sample sample weighting amount, unit is gram (g);
V---sample handling processes dilution volume, unit is milliliter (mL);
10---unit conversion factor.
2. a kind of detection method measuring Determination of galactooligosacchariin in formula milk as claimed in claim 1, is characterized in that, described galactose and lactose gas chromatograph-mass spectrometer (GCMS) measure.
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| CN105223062B (en) * | 2015-11-10 | 2020-09-29 | 黑龙江省乳品工业技术开发中心 | Method for purifying galactooligosaccharide in infant formula milk powder |
| CN105424858A (en) * | 2015-12-31 | 2016-03-23 | 广州甘蔗糖业研究所 | Efficient liquid chromatography tandem mass spectrometry detection method for galactooligosaccharides in milk powder |
| CN105424858B (en) * | 2015-12-31 | 2017-03-22 | 广州甘蔗糖业研究所 | Efficient liquid chromatography tandem mass spectrometry detection method for galactooligosaccharides in milk powder |
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