CN101034066A - Method for testing lactose hydrolysis ratio in low lactose milk - Google Patents
Method for testing lactose hydrolysis ratio in low lactose milk Download PDFInfo
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Abstract
This application discloses a glucose oxidase - spectrophotometric method of measuring low lactose creamery's lactose hydrolization rate. this method includes (1) determine lactose concentration after creamery hydrolization; (2) determine lactose concentration before creamery hydrolization;(3) calculate lactose hydrolization rate, hydrolyze rate =( lactose concentration after hydrolization / lactose concentration before hydrolization)X100%. This test method has feature of fast velocity, low cost, high accuracy.
Description
Technical field
The present invention relates to a kind of method of measuring lactose hydrolysis ratio in the low-lactose milk, more specifically, relate to the method for the lactose hydrolysis ratio that adopts the described glucose oxidase of the application-spectrophotometry low-lactose milk.
Background technology
Low-lactose milk is to utilize lactase that lactose in the milk is hydrolyzed, and hydrolysis produces glucose and galactose, so percent hydrolysis is an important production target weighing the low-lactose milk hydrolysis degree.It is several that the method for the mensuration low-lactose milk percent hydrolysis of being introduced in the present documents and materials report is mainly high performance liquid chromatography (HPLC) (Gan Binbin, 2001), solidifying point descent method (groom's rosy clouds, 2002), iodimetric titration (Qin Lihu, 2003) etc.Wherein the HPLC method be with chromatograph as the basis, its biggest advantage is exactly the accuracy height that lactose hydrolysis ratio is measured, but that weak point is the expense of each start is very high, even large-scale this method of dairy products enterprise can not be accepted; And other several method is also owing to requiredly have special-purpose instrument and a relevant matched reagent thereof, or owing to the excessive cycle of its check and analysis and the result's that measured reasons such as poor accuracy, also making these methods be difficult at present in the actual detection analysis is applied, and these methods that detect the low-lactose milk lactose hydrolysis ratios must have in the same old way, promptly must keep unhydrolysed milk, so that the concentration of lactose in the milk when being used for measuring not hydrolysis when measuring lactose hydrolysis ratio, this can't realize for finished product detection.
The inventor is by discovering, the hydrolysis under the effect of lactase of the lactose of 1 molecule generates the glucose of 1 molecule and the galactose of 1 molecule, glucose in the sample generates gluconic acid and hydrogen peroxide through the glucose oxidase oxydasis, the latter is under catalatic effect, and be condensed into than woods and phenol coupling can be by the red quinones of spectrophotometric determination by carrying with reductibility 4-ammonium aldehyde.The depth of the quinones color that generates is directly proportional with glucose content, the absorption photometric value of difference bioassay standard pipe and sample hose, then can calculate the content of glucose, can calculate the lactose concn of hydrolysis by concentration of glucose, if milk is complete hydrolysis, then can calculate the concentration of lactose in the former milk of hydrolysis by concentration of glucose.The inventor has finished the present invention based on this.
Summary of the invention
The invention solves the weak point in the prior art scheme that background technology mentions, essence from the lactose hydrolysis reaction, a kind of method-glucose oxidase-spectrophotometric method of measuring lactose hydrolysis ratio in the low-lactose milk is provided, it adopts the percent hydrolysis of lactose in glucose oxidase-spectrophotometry milk, can reduce the detection cost, measure the lactose hydrolysis ratio of low-lactose milk quickly and easily.
The technical scheme of glucose oxidase-spectrophotometric method of the present invention is as follows:
(a). the lactose concn after the hydrolysis of mensuration low-lactose milk;
(b). measure the preceding lactose concn of low-lactose milk hydrolysis;
(c).
The concrete operations step is as follows:
(a). the lactose concn after the hydrolysis of mensuration low-lactose milk:
Add the mix reagent protein precipitation in elder generation's giving lactose hydrolysed milk and prepare sample A; Then, draw isopyknic standard glucose solution and sample A, for example respectively get 20 μ L, join enzyme reagent and mix with phenol reagent in the working fluid of forming; React a period of time preferred 15-30 minute down at 30 ℃-40 ℃; In 500-520nm, the ultraviolet-visible pectrophotometer of preferred 505nm is measured its absorbance down, and according to standard glucose concentration, the concentration of glucose of calculation sample is calculated the concentration of the lactose of hydrolysis again according to this densimeter;
(b). measure the preceding lactose concn of low-lactose milk hydrolysis;
In the step (b), at first prepare lactose complete hydrolysis milk: add 5000-10000NLU (NeutralLactase Units in every liter of low-lactose milk, be neutral fcc lactase units) lactase, in 30 ℃-40 ℃ water-bath, react a period of time, preferred 2-4 hour, this amount and reaction time were guaranteed unhydrolysed lactose complete hydrolysis in the low-lactose milk---be lactose hydrolysis ratio can reach 100%; Then, add mix reagent, preparation sample B; Draw isopyknic standard glucose solution and sample B again, join enzyme reagent and mix with phenol reagent in the working fluid of forming; React a period of time preferred 15-30 minute down at 30 ℃-40 ℃; In 500-520nm, the ultraviolet-visible pectrophotometer of preferred 505nm is measured its absorbance down, and according to standard glucose concentration, the concentration of glucose of calculation sample is calculated the concentration of the lactose before the hydrolysis again according to this densimeter;
(c). calculate lactose hydrolysis ratio
In such scheme, described mix reagent is that reagent carrez first and carrez second are formed, and the czrrez first is 1.5%-3.0% by mass percent concentration, preferred 2.0% K
4Fe (CN)
63H
2The O aqueous solution is formed; Carrez second is 3.0%-6.0% by mass percent concentration, preferred 4.0% ZnSO
47H
2The O aqueous solution is formed, and preferably, described mix reagent is that isopyknic carrez first and carrez second are formed.
Wherein, in the preparation of sample A and sample B, preferably, also adding can be diluted to milk 10-100 doubly, and for example the distilled water of 20 times of volumes leaves standstill a period of time after the vibration afterwards again, preferred 10-20 minute, filters then.
Wherein, used working fluid is made up of enzyme reagent and phenol reagent, preferably, is made up of isopyknic enzyme reagent and isopyknic phenol reagent; Enzyme reagent by the glucose oxidase of 〉=10500U/L, 〉=peroxidase of 8000U/L and 〉=the amino antipyrine of 0.1g/L4-forms, phenol reagent is made up of the phenol of 〉=0.5g/L.
Wherein, concentration of glucose is 0-4.5g/L in the sample, and along with the increase of glucose, absorbance and the concentration of measuring pipe are linear dependence, r
2〉=0.995.
Wherein, the concentration of sample glucose (g/L) is:
1 is the concentration (g/L) of standard glucose; The lactose concn of hydrolysis (g/L) is concentration of glucose (g/L) * 2 * (10-100), wherein the lactose hydrolyzable of 1 unit mass of 2 expressions becomes the glucose of 2 unit masses, the extension rate of the original milk of getting when 10-100 representative preparation sample A and B, down together.
Wherein, lactose complete hydrolysis milk prepares by following method: give the lactase that adds 5000-10000NLU/L in the low-lactose milk again, hydrolysis 2-4 hour, for example add the lactase hydrolysis 2 hours of 10000NLU/L, with unhydrolysed lactose complete hydrolysis; If milk powder, after the milk powder reduction, add the lactase of 5000-10000NLU/L again, hydrolysis 2-4 hour, for example add the lactase hydrolysis 4 hours of 5000NLU/L, with unhydrolysed lactose complete hydrolysis.
In the present invention, described low-lactose milk is meant and adds lactase in the milk, makes the lactose in milk partial hydrolysis, the percent hydrolysis of lactose 〉=70%.General lactose in milk percent hydrolysis reaches 70%, just can solve lactose intolerance (groom's rosy clouds, 2002).
Compare with present other detection methods of the prior art, detection method speed of the present invention is fast, expense is low, accuracy is high, and the percent hydrolysis that detects the finished product low-lactose dairy product does not need control sample, described control sample is for must be with the milk of the same batch that does not add lactase, and method of the present invention is particularly suited for detecting the lactose hydrolysis ratio in the finished product.
Embodiment
Embodiment 1
A. measure low-lactose liquid milk lactose hydrolysis amount:
(1) preparation sample A: get 1ml lactose hydrolysis milk and add 1ml carrez reagent first and 1ml carrez reagent second protein precipitation, add 7ml distilled water diluting to 10 times again, left standstill after the vibration about 10 minutes, filter then, filtered fluid is called sample A;
(2) drawing the glucose standard solution of 20 μ L 1g/L and 20 μ L sample A respectively joins in enzyme reagent and the working fluid that the phenol reagent equal-volume mixes;
(3) afterwards, be placed in 30 ℃ of waters bath with thermostatic control, reacted 30 minutes, measure the absorbance of glucose titer and sample at 500nm wavelength place respectively, calculate the concentration of glucose in the filtrate:
The concentration of the glucose that the lactose that the concentration of this glucose is exactly hydrolysis discharges is so just can calculate the concentration of the lactose of hydrolysis by the concentration of glucose.
(4) lactose concn (the g/L)=concentration of glucose (g/L) * 2 * 10 of calculating hydrolysis.
B. measure lactose content in the preceding liquid milk of hydrolysis:
(1) preparation lactose complete hydrolysis milk: the lactase that in the lactose hydrolysis milk of 1000mL, adds 5000 units again, be that ultimate density is the lactase of 5000NLU/L, in 30 ℃ water-bath, placed 4 hours, guarantee the whole hydrolysis of unhydrolysed lactose in the lactose hydrolysis milk, promptly get lactose complete hydrolysis milk;
(2) preparation sample B: get 1mL lactose complete hydrolysis milk and add 1mL carrez reagent first and 1mL carrez reagent second protein precipitation, add 7mL distilled water diluting to 10 times again, left standstill after the vibration about 10 minutes, filter then;
(3) draw glucose standard solution and the 20 μ L sample B of 20 μ L 1g/L respectively, join and mix in the working fluid of forming with phenol reagent by isopyknic enzyme reagent;
(4) then, be placed in 30 ℃ of waters bath with thermostatic control, reacted 30 minutes, measure the absorbance of glucose titer and sample at 500nm wavelength place respectively, calculate the concentration of glucose in the filtrate then:
This concentration of glucose is exactly the amount of the glucose that produces after the lactose complete hydrolysis, so can extrapolate before the hydrolysis concentration of lactose in the milk by the concentration of glucose:
(5). calculate lactose concn in the preceding milk of hydrolysis:
Lactose concn (g/L)=concentration of glucose (g/L) * 2 * 10.
C. calculate lactose hydrolysis ratio
Embodiment 2
A. measure low-lactose liquid milk lactose hydrolysis amount:
(1) preparation sample A: get 10ml lactose hydrolysis milk and add 10ml carrez reagent first and 10ml carrez reagent second protein precipitation, add 970ml distilled water diluting to 100 times again, left standstill after the vibration about 20 minutes, filter then, filtered fluid is called sample A;
(2) drawing the glucose standard solution of 50 μ L 1g/L and 50 μ L sample A respectively joins by in enzyme reagent and the working fluid that the phenol reagent equal-volume mixes;
(3) afterwards, be placed in 40 ℃ of waters bath with thermostatic control, reacted 15 minutes, measure the absorbance of glucose titer and sample at 520nm wavelength place respectively, calculate the concentration of glucose in the filtrate:
The concentration of the glucose that the lactose that the concentration of this glucose is exactly hydrolysis discharges is so just can calculate the concentration of the lactose of hydrolysis by the concentration of glucose.
(4) lactose concn (the g/L)=concentration of glucose (g/L) * 2 * 100 of calculating hydrolysis.
B. measure lactose content in the preceding liquid milk of hydrolysis:
(1) preparation lactose complete hydrolysis milk: the lactase that in the lactose hydrolysis milk of 1000mL, adds 10000 units again, be that ultimate density is the lactase of 10000NLU/L, in 40 ℃ water-bath, placed 2 hours, guarantee the whole hydrolysis of unhydrolysed lactose in the lactose hydrolysis milk, promptly get lactose complete hydrolysis milk;
(2) preparation sample B: get 10mL lactose complete hydrolysis milk and add 10mL carrez reagent first and 10mL carrez reagent second protein precipitation, add 970mL distilled water diluting to 100 times again, left standstill after the vibration about 20 minutes, filter then;
(3) draw glucose standard solution and the 50 μ L sample B of 50 μ L 1g/L respectively, join and mix in the working fluid of forming with phenol reagent by isopyknic enzyme reagent;
(4) then, be placed in 40 ℃ of waters bath with thermostatic control, reacted 15 minutes, measure the absorbance of glucose titer and sample at 520nm wavelength place respectively, calculate the concentration of glucose in the filtrate then:
This concentration of glucose is exactly the amount of the glucose that produces after the lactose complete hydrolysis, so can extrapolate before the hydrolysis concentration of lactose in the milk by the concentration of glucose:
(5). calculate lactose concn in the preceding milk of hydrolysis:
Lactose concn (g/L)=concentration of glucose (g/L) * 2 * 100.
C. calculate lactose hydrolysis ratio
Embodiment 3
A. the mensuration of lactose hydrolysis amount after the low-lactose milk powder hydrolysis:
(1) preparation sample A: get the volumetric flask of 10g milk powder constant volume to 100mL, make reduction milk powder, get 5mL reduction milk powder then and add 5mL carrez reagent first and 5mL carrez reagent second protein precipitation, add 235mL distilled water diluting to 50 times again, left standstill after the vibration about 15 minutes, filter then, filtered fluid is sample A;
(2) glucose standard solution and the 30 μ L sample A that draw 30 μ L 1g/L respectively join in enzyme reagent and the phenol reagent equal-volume hybrid working liquid;
(3) afterwards, be placed in 35 ℃ of waters bath with thermostatic control, reacted 20 minutes, measure the absorbance of glucose titer and sample at 510nm wavelength place respectively, calculate the concentration of glucose in the filtrate:
The concentration of this glucose be exactly in the reduction milk powder hydrolysis the concentration of the glucose that discharges of lactose, so the concentration by glucose just can calculate and reduce the concentration of the lactose of hydrolysis in the milk powder.
(4) lactose concn of calculating hydrolysis:
Lactose concn (g/L)=concentration of glucose (g/L) * 2 * 50.
B. measure the preceding milk powder lactose content of hydrolysis:
(1) preparation lactose complete hydrolysis milk powder: get 10g milk powder constant volume and make reduction milk powder to the volumetric flask of 100mL, the lactase that adds 750 units then, be the lactase of 7500NLU/L finally promptly, in 35 ℃ water-bath, placed 3 hours, guarantee the whole hydrolysis of unhydrolysed lactose in the lactose hydrolysis milk powder, be lactose complete hydrolysis milk powder;
(2) preparation sample B: get 5mL lactose complete hydrolysis milk and add 5mL carrez reagent first and 5mL carrez reagent second protein precipitation, add 235mL distilled water diluting to 50 times again, left standstill after the vibration about 15 minutes, filter then, filtered fluid is sample B;
(3) draw glucose standard solution and the 30 μ L sample B of 30 μ L 1g/L respectively, join in enzyme reagent and the working fluid that the phenol reagent equal-volume mixes;
(4) afterwards, be placed in 35 ℃ of waters bath with thermostatic control, reacted 20 minutes, measure the absorbance of glucose titer and sample at 510nm wavelength place respectively, allow the back calculate the concentration of glucose in the filtrate:
This concentration of glucose is exactly the amount of the glucose that produces after the lactose complete hydrolysis in the reduction milk powder, so can extrapolate the content of lactose before the hydrolysis of reduction milk powder by the concentration of glucose.
(5) calculate the preceding lactose concn of hydrolysis
Lactose concn (g/L)=concentration of glucose (g/L) * 2 * 50.
C. calculate the milk powder lactose hydrolysis ratio:
Test 1
The recovery test of glucose oxidase-spectrophotometry glucose
Accuracy to glucose oxidase-spectrophotometry glucose content is tested, get 0.1018,0.1504,0.2021,0.2507 respectively, 0.3016g glucose joins in the 100mL milk, measure the value of glucose behind the method processing sample according to embodiment in the present specification 1, measured result listed in table 1:
The recovery test result of table 1 glucose oxidase-spectrophotometry glucose
| 1 | 2 | 3 | 4 | 5 |
| Amount (g/L) recovery (%) that records in the actual addition of sample absorbance (g/L) test | 0.247 1.018 0.988 97.05 | 0.363 1.504 1.425 96.54 | 0.508 2.021 2.032 100.54 | 0.620 2.507 2.480 98.92 | 0.736 3.016 2.944 97.61 |
As can be seen from Table 1, the recovery of glucose is between 97%-101%, thereby the accuracy that shows the glucose content that the application's method is measured is very high.
Test 2
The precision of lactose hydrolysis ratio test in glucose oxidase-spectrophotometry milk
Precision to lactose hydrolysis in glucose oxidase-spectrophotometry milk is tested, give the lactase that adds 2000NLU/L in milk hydrolysis 1 hour under 37 ℃ condition, after the hydrolysis in boiling water with the lactase deactivation, measure the amount of the lactose of lactose hydrolysis front and back behind the method processing sample according to described embodiment 1 of the application or embodiment 2, measured result is as shown in table 2:
The precision of lactose hydrolysis ratio test in table 2 glucose oxidase-spectrophotometry milk
| 1 | 2 | 3 | 4 | 5 | 6 | Mean value | Standard deviation | The coefficient of variation | |
| The content of lactose content (g/100mL) the complete hydrolysis sample absorbance lactose complete hydrolysis that has been hydrolyzed in the sample absorbance milk of hydrolysis is concentration (g/100mL) lactose hydrolysis ratio (%) of lactose before the milk hydrolysis | 0.334 2.672 0.618 4.944 | 0.339 2.712 0.651 5.208 | 0.344 2.752 0.647 5.176 | 0.351 2.808 0.645 5.160 | 0.346 2.768 0.654 5.232 53.13 | 0.342 2.736 0.654 5.232 | - 2.741 - 5.159 | - 0.047 - 0.109 | - 1.7％ - 2.1％ |
Annotate: the absorbance of standard pipe is: 0.250.
Can find out that from table 2 coefficient of variation of measurement result is respectively 1.7% and 2.1%, thereby shows, the precision of lactose hydrolysis ratio is fine in the milk that the application's method is measured.
The described in this article and restriction that is not subjected to particular disclosed herein that requires of the present invention is because embodiment is intended to set forth some aspect of the present invention.Equal embodiment is also included within the present invention arbitrarily.In fact, except show herein with describe, to those skilled in the art, the present invention is conspicuous to the various changes of foregoing description.These changes also fall in the scope of accessory claim.
Claims (10)
1. the assay method of lactose hydrolysis ratio in the low-lactose milk is characterized in that this method comprises:
(a). the lactose concn after the hydrolysis of mensuration low-lactose milk;
(b). measure the preceding lactose concn of low-lactose milk hydrolysis;
(c). calculate lactose hydrolysis ratio
Wherein,
In the step (a), at first add the mix reagent protein precipitation in the giving lactose hydrolysed milk and prepare sample A; Then, draw isopyknic standard glucose solution and sample A, join and mix in the working fluid of forming with phenol reagent by enzyme reagent; React a period of time down at 30 ℃-40 ℃; Measure its absorbance down in the ultraviolet-visible pectrophotometer of 500-520nm, according to standard glucose concentration, the concentration of glucose of calculation sample is calculated the lactose concn of hydrolysis again according to this densimeter;
In the step (b), at first prepare lactose complete hydrolysis milk: the lactase that adds 5000-10000NLU in every liter of low-lactose milk, react a period of time in 30 ℃-40 ℃ water-bath, this amount and reaction time are guaranteed unhydrolysed lactose complete hydrolysis in the low-lactose milk; Then, add mix reagent, preparation sample B; Draw isopyknic standard glucose solution and sample B again, join enzyme reagent and mix with phenol reagent in the working fluid of forming; React a period of time down at 30 ℃-40 ℃; Measure its absorbance down in the ultraviolet-visible pectrophotometer of 500-520nm, according to standard glucose concentration, the concentration of glucose of calculation sample is calculated the concentration of the lactose before the hydrolysis again according to this densimeter.
2. assay method according to claim 1 is characterized in that, wherein said mix reagent is that reagent carrez first and carrez second are formed, and the czrrez first is the K of 1.5-3.0% by mass percent concentration
4Fe (CN)
63H
2The O aqueous solution is formed; Carrez second is the ZnSO of 3.0%-6.0% by mass percent concentration
47H
2The O aqueous solution is formed.
3. according to the assay method of claim 1 or 2, it is characterized in that, wherein in step (a) with (b), with the working fluid reaction time be 15-30 minute, in step (b), with the lactase reaction time be 2-4 hour, the czrrez first is 2.0% K by mass percent concentration
4Fe (CN)
63H
2The O aqueous solution is formed; Carrez second is 4.0% ZnSO by mass percent concentration
47H
2The O aqueous solution is formed.
4. assay method according to claim 3 is characterized in that, wherein in the preparation of sample A and sample B, after adding mix reagent, also can add the distilled water that milk can be diluted to 10-100 times of volume, leave standstill 10-20 minute after the vibration more afterwards, filter then.
5. assay method according to claim 4, it is characterized in that, wherein the concentration of standard glucose solution is 1g/L, and described mix reagent is that isopyknic carrez first and carrez second are formed, and wherein absorbance is to measure under the ultraviolet-visible pectrophotometer of 500-520nm.
6. assay method according to claim 5 is characterized in that, wherein used working fluid is made up of isopyknic enzyme reagent and isopyknic phenol reagent.
7. according to claims 6 described assay methods, it is characterized in that, wherein enzyme reagent by the glucose oxidase of 〉=10500U/L, 〉=peroxidase of 8000U/L and 〉=the amino antipyrine of 0.1g/L4-forms, wherein phenol reagent is made up of the phenol of 〉=0.5g/L.
8. assay method according to claim 1 is characterized in that, wherein concentration of glucose is in the sample:
Numerical value is 0-4.5g/L, and along with the increase of concentration of glucose, absorbance and the concentration of measuring pipe are linear dependence, r
2〉=0.995.
9. according to the assay method described in the claim 1, it is characterized in that wherein the lactose concn of hydrolysis (g/L) is concentration of glucose (g/L) * 2 * (10-100).
10. according to claim 1 or 7 described assay methods, it is characterized in that, wherein lactose complete hydrolysis milk prepares by following method: give the lactase that adds 5000-10000NLU/L in the low-lactose milk again, with unhydrolysed lactose complete hydrolysis, if milk powder, after the milk powder reduction, add the lactase hydrolysis of 5000-10000NLU/L again.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102445517A (en) * | 2011-09-19 | 2012-05-09 | 安徽达诺乳业有限公司 | Sucrose detection method |
| CN104122250A (en) * | 2014-07-04 | 2014-10-29 | 华东理工大学 | Method for rapid detection of lactose in milk |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100441099C (en) * | 2003-12-22 | 2008-12-10 | 北京三元食品股份有限公司 | Method for inhibiting brown stain of low lactose milk and products thereof |
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2007
- 2007-02-03 CN CN2007100071454A patent/CN101034066B/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102445517A (en) * | 2011-09-19 | 2012-05-09 | 安徽达诺乳业有限公司 | Sucrose detection method |
| CN104122250A (en) * | 2014-07-04 | 2014-10-29 | 华东理工大学 | Method for rapid detection of lactose in milk |
| CN104122250B (en) * | 2014-07-04 | 2019-03-26 | 华东理工大学 | A kind of method of quick detection lactose |
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