CN102621261A - Detection method for nucleotide content in infant cereal - Google Patents

Detection method for nucleotide content in infant cereal Download PDF

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Publication number
CN102621261A
CN102621261A CN2012100292212A CN201210029221A CN102621261A CN 102621261 A CN102621261 A CN 102621261A CN 2012100292212 A CN2012100292212 A CN 2012100292212A CN 201210029221 A CN201210029221 A CN 201210029221A CN 102621261 A CN102621261 A CN 102621261A
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China
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sample
nucleotide
100ml
standard
ultrapure water
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CN2012100292212A
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Chinese (zh)
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黄树杰
林旭彬
陈润汉
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广东东泰乳业有限公司
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Priority to CN2012100292212A priority Critical patent/CN102621261A/en
Publication of CN102621261A publication Critical patent/CN102621261A/en

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Abstract

The present invention relates to a detection method for nucleotide content in infant cereal. The method comprises the following steps: sample enzymolysis, ultrasonic extraction, high-speed centrifugation, purification and filtration, preparation of chromatography conditions and quantitative analysis, wherein the sample enzymolysis step comprises: weighing 5-10 g of the sample, placing in a 100-200 mL flask, adding 0.2-0.4 g of taka-diastase, 0.2-0.4 g of papain and 30-60 mL of water with a temperature of 40-50 DEG, uniformly stirring, and placing in an incubator with a temperature of 50 DEG C to culture for 2-4 hours, the ultrasonic extraction step comprises: placing the digested sample on an ultrasonic oscillator to carry out an ultrasonic treatment, adjusting the pH value with an acetic acid solution, and carrying out volume metering with ultrapure water, the high-speed centrifugation, purification and filtration step comprises: centrifugating the sample solution with a centrifuge, filtering the resulting supernatant with medium speed filter paper, filtering with a water system filtration membrane with an aperture of 0.45 mum to provide for HPLC determination, and carrying out quantitative analysis with an external standard method. According to the present invention, the nucleotide content in the infant cereal is detected, and advantages of convenience, rapidness, accuracy and the like are provided.

Description

Nucleotide detection method of content in infant's ground rice

Technical field

The present invention relates to nucleotide content detecting method in a kind of infant's ground rice.

Background technology

Nucleotide comprises cytidylic acid, adenylic acid, uridylate, guanylic acid and inosinic acid; Abbreviate CMP, AMP, UMP, GMP and IMP respectively as; Nucleotide can be kept immune normal function, improves the resistibility of human body to all kinds of germs.Nucleotide can promote small intestine ripe, improves intestinal absorption ability.Can also stimulation of bifidobacteria grow, reduce the generation of baby's constipation, diarrhoea.Nucleotide also has fabulous antioxidation in addition, keeps the normal function of liver.

Just added nucleotide in the new infant's rice flour product released of company at present,, corresponding detecting method has not been arranged as yet in the GB for the assay of nucleotide in the food.Therefore, the detection method of content of nucleotide is suitable for the required nucleotide ground rice that contains of infant growth most significance is arranged mixing in the exploitation infant ground rice.Because of Gu Jizhong contains more starch and vegetable protein, in the pasty state, cause easily and disturb and filtration difficulty after the sample dissolution, therefore add Taka-diastase and papain and carry out enzymolysis solution existing problems.For the nucleotide in the paddy basic pattern article is fully extracted, this method adopts ultrasonic Extraction, carries out centrifugal purification again.Reduce the loss to greatest extent and disturb, improved the accuracy of experiment feasibility and testing result.

Summary of the invention

The object of the present invention is to provide nucleotide content detecting method in a kind of infant's ground rice.Be intended to convenient, fast, detect the content of nucleotide in infant's ground rice exactly.

The present invention, its principle is: the starch in infant's ground rice and vegetable protein earlier with behind Taka-diastase and the papain enzymolysis, are utilized Extraction by Ultrasound, the high speed centrifugation purification, through UV-detector mensuration, external standard method is quantitative.

The present invention comprises following job step:

(1) reagent is prepared, and agents useful for same comprises:

(a) methyl alcohol: chromatographically pure;

(b) 4-butyl ammonium hydrogen sulfate: chromatographically pure;

(c) dipotassium hydrogen phosphate: analyze pure;

(d) potassium dihydrogen phosphate: chromatographically pure;

(e) acetate: top grade is pure;

(f) papain: enzyme activity >=600U/g, 0.2-0.4g;

(g) Taka-diastase: enzyme activity >=4000U/g, 0.2-0.4g;

(h) nucleotide standard items comprise CMP, AMP, UMP, GMP and IMP: purity >=98%;

(i) 0.1mol/L potassium dihydrogen phosphate: take by weighing the 13.60g potassium dihydrogen phosphate with ultrapure water dissolving and be settled to 1000mL;

(j) 0.1mol/L dipotassium hydrogen phosphate solution: take by weighing the 2.28g dipotassium hydrogen phosphate with ultrapure water dissolving and be settled to 100mL;

(k) 100mL/L acetic acid solution: draw 10mL acetate, add ultrapure water and be settled to 100mL;

(l) contain the 0.01mol/L potassium phosphate buffer of 1.40mmol/L 4-butyl ammonium hydrogen sulfate: get the 100mL0.1mol/L potassium dihydrogen phosphate; Add the 0.4753g 4-butyl ammonium hydrogen sulfate; Transfer pH to 3.3 with the 0.1mol/L dipotassium hydrogen phosphate solution, with the ultrapure water constant volume to 1000mL;

(m) nucleotide standard mixed liquor: accurately take by weighing nucleotide standard items CMP, AMP, UMP, GMP, each 10-20mg of IMP respectively; The mixing back is with the ultrapure water dissolving and be settled to 100ml, and the CMP in this standard mixed liquor, AMP, UMP, GMP, IMP content respectively are 100-200 μ g/mL;

(n) nucleotide standard operation liquid: draw standard mixed liquor 2mL, 4mL, 6mL, 8mL, 10mL respectively in 5 100ml volumetric flasks, add ultrapure water and be settled to 100mL, be made into nucleotide series standard working fluid;

(2) instrument and equipment, instrument and equipment comprises:

(a) high performance liquid chromatograph: Tianjin, island LC-20AT pump, SPD-20A UV-detector;

(b) electronic balance: be accurate to 0.0001g;

(c) Extraction by Ultrasound device;

(d) supercentrifuge;

(3) sample preparation:

(a) enzymolysis: take by weighing sample 5g-10g, be accurate to 1mg, place the 100mL-200mL triangular flask, add the 0.2-0.4g Taka-diastase, the 0.2-0.4g papain adds 30-60mL40-50 ℃ of water, stirs, and puts in 50 ℃ of incubators and cultivates 2-4h;

(b) Extraction by Ultrasound: the sample behind the enzymolysis is placed ultrasonic Extraction 10-20min on the ultrasonic oscillator, take out and be cooled to room temperature, transfer pH to 3.3, sample is moved into the 100mL volumetric flask, use the ultrapure water constant volume with the 100mL/L acetic acid solution;

(c) high speed centrifugation purification filtering: appearance liquid changes the 50mL centrifuge tube in the centrifugal 5min of the hydro-extractor of 10000r/min behind the constant volume; Supernatant is through the middling speed filter paper filtering; Draw filtrating 1-2mL through 0.45 μ m water system membrane filtration with disposable syringe, filtrating is final samples liquid, supplies HPLC to measure.Retention time is qualitative, and external standard method is quantitative;

(4) chromatographic condition, chromatographic condition comprises:

(a) chromatographic column: CAPCELL PAK C18 (250 mm * 4.6mm, 5 μ m) or suitable person;

(b) sample size: 10 μ L;

(c) flow velocity: 1.0mL/min;

(d) UV-detector wavelength: 254nm;

(e) column temperature: 25 ℃;

(f) moving phase: 4% methyl alcohol-96% phosphate buffer, low pressure concentration wash-out;

(5) quantitative test:

(a) typical curve is drawn: the standard series working fluid being injected high performance liquid chromatograph respectively, measure corresponding peak area, is ordinate with the peak area, and standard operation liquid concentration is horizontal ordinate drawing standard curve;

(b) test solution is measured: the appearance liquid that will prepare injects high performance liquid chromatograph, records corresponding peak area, with typical curve sample is carried out quantitatively recording test solution concentration;

(c) calculate, computing formula is: X=C * V 1* 100/ (m * 1000), in the formula:

X: each components contents of nucleotide in the sample, unit is mg/100g;

C: by the test solution concentration that typical curve obtains, unit is μ g/mL;

V 1: sample constant volume, unit are mL;

M: sample mass, unit are g;

Nucleotide content Z=X in the sample CMP+ X AMP+ X UMP+ X GMP+ X IMP

This method moving phase is 4% methyl alcohol-96% phosphate buffer, and pH is 3.3.Moving phase ratio and pH value are very big to experimental result influence, thus must strict control, and typical curve and moving phase must be prepared and use on the same day.

The present invention, the content of detection nucleotide has advantages such as convenient, fast, accurate in infant's ground rice.Be suitable for most the required nucleotide ground rice that contains of infant growth significance is arranged mixing.

Description of drawings

Fig. 1 is a nucleotide content standard colors spectrogram; Fig. 2 is the sample oligonucleotide chromatogram; Fig. 3 is the canonical plotting of CMP peak area and concentration; Fig. 4 is the canonical plotting of AMP peak area and concentration; Fig. 5 is the canonical plotting of UMP peak area and concentration; Fig. 6 is the canonical plotting of GMP peak area and concentration; Fig. 7 is the canonical plotting of IMP peak area and concentration; Among the figure, horizontal ordinate is a standard operation liquid concentration, and ordinate is the corresponding peak area of standard operation liquid.In Fig. 1 and Fig. 2, peak sequence is confirmed as CMP, AMP, UMP, GMP, IMP with nucleotide standard substance list mark sample introduction, and retention time is qualitative, and external standard method is quantitative.

Embodiment

The detection method of content of nucleotide in a kind of infant's ground rice comprises following job step:

(1) reagent is prepared, and agents useful for same comprises:

(a) methyl alcohol: chromatographically pure;

(b) 4-butyl ammonium hydrogen sulfate: chromatographically pure;

(c) dipotassium hydrogen phosphate: analyze pure;

(d) potassium dihydrogen phosphate: chromatographically pure;

(e) acetate: top grade is pure;

(f) papain: enzyme activity >=600U/g, 0.2g;

(g) Taka-diastase: enzyme activity >=4000U/g, 0.2g;

(h) nucleotide standard items comprise CMP, AMP, UMP, GMP and IMP: purity >=98%;

(i) 0.1mol/L potassium dihydrogen phosphate: take by weighing the 13.60g potassium dihydrogen phosphate with ultrapure water dissolving and be settled to 1000mL;

(j) 0.1mol/L dipotassium hydrogen phosphate solution: take by weighing the 2.28g dipotassium hydrogen phosphate with ultrapure water dissolving and be settled to 100mL;

(k) 100mL/L acetic acid solution: draw 10mL acetate, add ultrapure water and be settled to 100mL;

(l) contain the 0.01mol/L potassium phosphate buffer of 1.40mmol/L 4-butyl ammonium hydrogen sulfate: get the 100mL0.1mol/L potassium dihydrogen phosphate; Add the 0.4753g 4-butyl ammonium hydrogen sulfate; Transfer pH to 3.3 with the 0.1mol/L dipotassium hydrogen phosphate solution, with the ultrapure water constant volume to 1000mL;

(m) nucleotide standard mixed liquor: accurately take by weighing nucleotide standard items CMP10mg, AMP20mg, UMP10mg, GMP10mg and IMP10mg respectively; The mixing back is with the ultrapure water dissolving and be settled to 100ml, and in this standard mixed liquor, CMP content is 100 μ g/mL; AMP content is 200 μ g/mL; UMP content is 100 μ g/mL, and GMP content is 100 μ g/mL, and IMP content is 100 μ g/mL;

(n) nucleotide standard operation liquid: draw standard mixed liquor 2mL, 4mL, 6mL, 8mL, 10mL respectively in 5 100ml volumetric flasks, add ultrapure water and be settled to 100mL, be made into nucleotide series standard working fluid;

(2) instrument and equipment, instrument and equipment comprises:

(a) high performance liquid chromatograph: Tianjin, island LC-20AT pump, SPD-20A UV-detector;

(b) electronic balance: be accurate to 0.0001g;

(c) Extraction by Ultrasound device;

(d) supercentrifuge;

(3) sample preparation:

(d) enzymolysis: take by weighing sample 5g, be accurate to 1mg, place the 100mL triangular flask, add the 0.2g Taka-diastase, the 0.2g papain adds 30mL40-50 ℃ of water, stirs, and puts in 50 ℃ of incubators and cultivates 2-4h;

(e) Extraction by Ultrasound: the sample behind the enzymolysis is placed ultrasonic Extraction 10-20min on the ultrasonic oscillator, take out and be cooled to room temperature, transfer pH to 3.3, sample is moved into the 100mL volumetric flask, use the ultrapure water constant volume with the 100mL/L acetic acid solution;

(f) high speed centrifugation purification filtering: appearance liquid changes the 50mL centrifuge tube in the centrifugal 5min of the hydro-extractor of 10000r/min behind the constant volume; Supernatant is through the middling speed filter paper filtering; Draw filtrating 1-2mL through 0.45 μ m water system membrane filtration with disposable syringe, filtrating is final samples liquid, supplies HPLC to measure.Retention time is qualitative, and external standard method is quantitative;

(4) chromatographic condition, chromatographic condition comprises:

(a) chromatographic column: CAPCELL PAK C18 (250 mm * 4.6mm, 5 μ m) or suitable person;

(b) sample size: 10 μ L;

(c) flow velocity: 1.0mL/min;

(d) UV-detector wavelength: 254nm;

(e) column temperature: 25 ℃;

(f) moving phase: 4% methyl alcohol-96% phosphate buffer, low pressure concentration wash-out;

(5) quantitative test:

(a) typical curve is drawn: the standard series working fluid being injected high performance liquid chromatograph respectively, measure corresponding peak area, is ordinate with the peak area, and standard operation liquid concentration is horizontal ordinate drawing standard curve, like Fig. 3-shown in Figure 7.When utilizing high performance liquid chromatograph to carry out determination and analysis, can directly read the numerical value of peak area;

(b) test solution is measured: the appearance liquid that will prepare injects high performance liquid chromatograph; Record corresponding peak area; With Fig. 3-Fig. 7 typical curve sample is carried out quantitatively, the concentration that records UMP in kind liquid is that the concentration of 1.907 μ g/mL, AMP is that the concentration of 0.724ug/ml, GMP is that the concentration of 4.904ug/ml, IMP is that 1.056ug/ml, CMP content do not detect;

(c) according to formula X=C * V 1* 100/ (m * 1000) can calculate: the content of UMP is that 3.80mg/100g, AMP content are that 1.44mg/100g, GMP content are that 9.76mg/100g, IMP content are that 2.10mg/100g, CMP content do not detect in the sample;

ID# Title Retention time Peak area Concentration Unit 1 CMP Do not detect ---- ---- μg/mL 2 UMP 5.798 37276 1.907 μg/mL 3 AMP 19.020 5238 0.724 μg/mL 4 GMP 20.784 119079 4.094 μg/mL 5 IMP 25.878 9329 1.056 μg/mL

(d) the nucleotide total content is in the sample:

Z=X CMP+X AMP+X UMP+X GMP+X IMP

=0+3.80+1.44+9.76+2.10

=17.1mg/100g;

(e) this case study on implementation detects this batch infant ground rice, and the nucleotide total content is 17.1mg/100g in the sample;

(6) recovery is calculated:

Get blank matrix solution, add variable concentrations nucleotide hybrid standard liquid respectively, handle by (3)-(5), sample introduction obtains the recovery (please see the following form) preferably:

(7) conclusion:

This method utilizes high performance liquid chromatograph simultaneously the content of 5 kinds of nucleotide in infant's ground rice to be detected; Need not repeat samples handles; Can measure the content of nucleotide in infant's ground rice fast and accurately; Be suitable for most the required nucleotide ground rice that contains of infant growth significance is arranged mixing, have higher utility.

Claims (2)

1. the detection method of content of nucleotide in infant's ground rice comprises sample enzymolysis, Extraction by Ultrasound, high speed centrifugation purification filtering, chromatographic condition and quantitative test, it is characterized in that comprising following job step:
(1) enzymolysis: take by weighing sample 5g-10g, be accurate to 1mg, place the 100mL-200mL triangular flask, add the 0.2-0.4g Taka-diastase, the 0.2-0.4g papain adds 30-60mL40-50 ℃ of water, stirs, and puts in 50 ℃ of incubators and cultivates 2-4h;
(2) Extraction by Ultrasound: the sample behind the enzymolysis is placed ultrasonic 10-20min on the ultrasonic oscillator, take out and be cooled to room temperature, transfer pH to 3.3, sample is moved into the 100mL volumetric flask, use the ultrapure water constant volume with the 100mL/L acetic acid solution;
(3) high speed centrifugation purification filtering: appearance liquid changes the 50mL centrifuge tube in the centrifugal 5min of the hydro-extractor of 10000r/min behind the constant volume, and supernatant is drawn filtrating 1-2mL through 0.45 μ m water system membrane filtration through the middling speed filter paper filtering with disposable syringe; Supply HPLC to measure, external standard method is quantitative.
2. the detection method of content of nucleotide in infant's ground rice comprises following job step:
(1) reagent is prepared, and agents useful for same comprises:
(a) methyl alcohol: chromatographically pure;
(b) 4-butyl ammonium hydrogen sulfate: chromatographically pure;
(c) dipotassium hydrogen phosphate: analyze pure;
(d) potassium dihydrogen phosphate: chromatographically pure;
(e) acetate: top grade is pure;
(f) papain: enzyme activity >=600U/g, 0.2-0.4g;
(g) Taka-diastase: enzyme activity >=4000U/g, 0.2-0.4g;
(h) nucleotide standard items comprise CMP, AMP, UMP, GMP and IMP: purity >=98%;
(i) 0.1mol/L potassium dihydrogen phosphate: take by weighing the 13.60g potassium dihydrogen phosphate with ultrapure water dissolving and be settled to 1000mL;
(j) 0.1mol/L dipotassium hydrogen phosphate solution: take by weighing the 2.28g dipotassium hydrogen phosphate with ultrapure water dissolving and be settled to 100mL;
(k) 100mL/L acetic acid solution: draw 10mL acetate, add ultrapure water and be settled to 100mL;
(l) contain the 0.01mol/L potassium phosphate buffer of 1.40mmol/L 4-butyl ammonium hydrogen sulfate: get the 100mL0.1mol/L potassium dihydrogen phosphate; Add the 0.4753g 4-butyl ammonium hydrogen sulfate; Transfer pH to 3.3 with the 0.1mol/L dipotassium hydrogen phosphate solution, with the ultrapure water constant volume to 1000mL;
(m) nucleotide standard mixed liquor: accurately take by weighing nucleotide standard items CMP, AMP, UMP, GMP, each 10-20mg of IMP respectively; The mixing back is with the ultrapure water dissolving and be settled to 100ml, and the CMP in this standard mixed liquor, AMP, UMP, GMP, IMP content respectively are 100-200 μ g/mL;
(n) nucleotide standard operation liquid: draw standard mixed liquor 2mL, 4mL, 6mL, 8mL, 10mL respectively in 5 100ml volumetric flasks, add ultrapure water and be settled to 100mL, be made into nucleotide series standard working fluid;
(2) instrument and equipment, instrument and equipment comprises:
(a) high performance liquid chromatograph: Tianjin, island LC-20AT pump, SPD-20A UV-detector;
(b) electronic balance: be accurate to 0.0001g;
(c) Extraction by Ultrasound device;
(d) supercentrifuge;
(3) sample preparation:
(a) enzymolysis: take by weighing sample 5g-10g, be accurate to 1mg, place the 100mL-200mL triangular flask, add the 0.2-0.4g Taka-diastase, the 0.2-0.4g papain adds 30-60mL40-50 ℃ of water, stirs, and puts in 50 ℃ of incubators and cultivates 2-4h;
(b) Extraction by Ultrasound: the sample behind the enzymolysis is placed ultrasonic Extraction 10-20min on the ultrasonic oscillator, take out and be cooled to room temperature, transfer pH to 3.3, sample is moved into the 100mL volumetric flask, use the ultrapure water constant volume with the 100mL/L acetic acid solution;
(c) high speed centrifugation purification filtering: appearance liquid changes the 50mL centrifuge tube in the centrifugal 5min of the hydro-extractor of 10000r/min behind the constant volume; Supernatant is through the middling speed filter paper filtering; Draw filtrating 1-2mL through 0.45 μ m water system membrane filtration with disposable syringe, filtrating is final samples liquid, supplies HPLC to measure; Retention time is qualitative, and external standard method is quantitative;
(4) chromatographic condition, chromatographic condition comprises:
(a) chromatographic column: CAPCELL PAK C18 (250 mm * 4.6mm, 5 μ m) or suitable person;
(b) sample size: 10 μ L;
(c) flow velocity: 1.0mL/min;
(d) UV-detector wavelength: 254nm;
(e) column temperature: 25 ℃;
(f) moving phase: 4% methyl alcohol-96% phosphate buffer, low pressure concentration wash-out;
(5) quantitative test:
(a) typical curve is drawn: the standard series working fluid being injected high performance liquid chromatograph respectively, measure corresponding peak area, is ordinate with the peak area, and standard operation liquid concentration is horizontal ordinate drawing standard curve;
(b) test solution is measured: the appearance liquid that will prepare injects high performance liquid chromatograph, records corresponding peak area, with typical curve sample is carried out quantitatively recording test solution concentration;
(c) calculate, computing formula is: X=C * V 1* 100/ (m * 1000), in the formula:
X: each components contents of nucleotide in the sample, unit is mg/100g;
C: by the test solution concentration that typical curve obtains, unit is μ g/mL;
V 1: sample constant volume, unit are mL;
M: sample mass, unit are g;
Nucleotide content Z=X in the sample CMP+ X AMP+ X UMP+ X GMP+ X IMP
CN2012100292212A 2012-02-09 2012-02-09 Detection method for nucleotide content in infant cereal CN102621261A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106226414A (en) * 2016-07-08 2016-12-14 黑龙江东方学院 Utilize the method that HPLC method measures baby formula milk powder nucleotide

Citations (3)

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Publication number Priority date Publication date Assignee Title
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CA2020831C (en) * 1989-07-11 2000-06-27 Gabor Puski Soluble rice protein concentrate
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Publication number Priority date Publication date Assignee Title
CA2020831C (en) * 1989-07-11 2000-06-27 Gabor Puski Soluble rice protein concentrate
CN1053731A (en) * 1990-12-24 1991-08-14 北京大学 Preparation method of a predigested high protein vegetable milk power substitate for babies
CN1358863A (en) * 2001-08-03 2002-07-17 华南理工大学 Improved method for rice enzyme method making maltose by enzyme pretreating

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D. S. KIM ET AL: "Development and Characterization of a Flavoring Agent from Oyster Cooker Effluent", 《J. AGRIC. FOOD CHEM.》 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106226414A (en) * 2016-07-08 2016-12-14 黑龙江东方学院 Utilize the method that HPLC method measures baby formula milk powder nucleotide

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Application publication date: 20120801