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534 2008, Vol. 29, No. 08 food science ÿRiddle

Study on the determination of reducing sugar content using 3,5-

dinitrosalicylic acid colorimetric method

Zhao Kai, Xu Pengju, Gu Guangye (School

of Food Engineering, Harbin University of Commerce, Provincial Key Laboratory of Food Science and Engineering, Harbin, Heilongjiang 150076)

Abstract: The factors affecting the determination of reducing sugar content using two DNS reagents were compared. The effects of reagent addition amount, color development time, measurement wavelength and storage time

after color development on the measurement results were discussed. The results show that the dosage of DNS reagent is 1.5ml, the color development time in boiling water bath is 5 minutes, and after the color development is

fixed to volume for 30 minutes, the data are measured under the wavelength conditions of 540nm and 550nm respectively, and the accuracy

and stability of the data are good. Keywords: 3,5-dinitrosalicylic acid (DNS); reducing sugar; colorimetric method; measurement conditions

Study on Determination of Reducing Sugar Content Using 3,5-Dinitrosalicylic Acid Method

ZHAO Kai, XUE Peng-ju, GU Guang-ye

(Province Key Laboratory of Food Science and Engineering, College of Food Engineering, Harbin University of

Commerce, Harbin 150076, China)

Abstract: Two kinds of 3, 5-dinitrosalicylic acid (DNS) agents were respectively used for determination of reducing sugar content, and effects on determination

results by the factors, such as the amount of DNS reagent developing time, wavelength and storage time were discussed. The results showed that on the

conditions of reagent amount around 1.5 ml, boiling bath time not less than 5 minutes, and standing for 30 minutes after developing, at the wavelength of 540

nm and 550 nm for reagent one and two respectively, the accurate results can be acquired.

Key words: 3, 5-dinitrosalicylic acid (DNS); reducing sugar; spectrophotometry; determination factor CLC number: TS231 Document

identification code: A Article number: 1002-6630(2008)08-0534-03

The 3,5-dinitrosalicylic acid (DNS) colorimetric method is a commonly used method for the Pullulanase (enzyme activity 1000ASPu/g) American Genencor Company; 3,5-dinitrosalicylic

determination of reducing sugars. This method is simple, fast, and highly sensitive. At the same time, acid, sodium hydroxide, glycerol, phenol, sodium sulfite, potassium sodium tartrate, glucose, sodium

by measuring the reducing power, this method can also be used to determine the debranching effect acetate, ice Acetic acid, the above drugs are of analytical grade. 1.2 Instruments and equipment

of pullulanase and other debranching enzymes on starch. However, when using this method for SP-721E visible

measurement, the measurement conditions introduced in different literatures are quite different [1-5], spectrophotometer Shanghai

thus affecting the accuracy and comparability of the measurement results. This experiment discusses Spectral Instrument Co., Ltd.; Acculab precision electronic balance; 702-type electric heating

the determination conditions of the DNS colorimetric method and points out the key factors affecting blast box Dalian Drying Oven Factory; DZF-6050 vacuum drying oven Shanghai Boxun Industrial

the analysis and determination, so as to make the determination results more accurate and Co., Ltd. Medical Equipment Factory ; THZ-82A water bath constant temperature oscillator Jiangsu

comparable, so as to facilitate the reference application when actually using this method for related Ronghua Instrument Manufacturing Co., Ltd.; 80-2 centrifuge Shanghai Pudong Physical and Optical

analysis and determination. Instrument Factory; HH-4 digital display constant temperature water bath Guohua Electric Co., Ltd.

1.3 Method
1 Materials and methods

1.1 Materials and reagents 1.3.1 DNS reagent preparation

Corn starch Heilongjiang Longfeng Corn Development Co., Ltd. Reagent 1: Weigh 3.25g of 3,5-dinitrosalicylic acid and dissolve it in a small amount of water

Received date: 200-04-25 Fund

projects: Heilongjiang Province “Eleventh Five-Year Plan” major research project (GA06B401-4); Heilongjiang Provincial Department of Education project (160204);

Heilongjiang Province Postdoctoral Fund Project (LBH-Z05037)

About the author: Zhao Kai (1974-), male, associate professor, Ph.D., research direction is starch chemistry and processing. E-mail: [email protected]
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ÿRiddle food science 2008, Vol. 29, No. 08 535

, transfer to a 500ml volumetric flask, add 162.5ml of 2mol/L sodium hydroxide solution, then DNS reagent, mix well and make a blank, heat in boiling water bath for 5 minutes, cool with running water

add 22.5g of glycerol, shake well, dilute to 500ml, store in a brown bottle in the refrigerator for and dilute to volume. Use the same conditions without adding glucose standard solution as a blank solution,

later use [1]. and measure the absorbance at a wavelength of 540 nm.

Reagent 2: Weigh 2.5g of 3,5-dinitrosalicylic acid and dissolve it in a small amount of

2 Results and analysis
water. Add 0.5g of phenol, then dissolve 0.075g of sodium sulfite, 2.5g of sodium hydroxide

and 50g of sodium potassium tartrate. Transfer it to a 500ml volumetric flask and shake. Mix 2.1 Selection of measurement wavelength

evenly, adjust the volume to 500ml, store in a brown bottle in the refrigerator until use [2].
The determination principle of the DNS method, that is, the 3,5-dinitrosalicylic acid colorimetric

1.3.2 Preparation of glucose standard solution method, is the reduction of 3,5-dinitrosalicylic acid and polysaccharide hydrolysis under neutral or alkaline

Weigh more than 1g of glucose and place it in a hot air drying oven at 98°C to dry to conditions. After the sugar is heated together, it is reduced to a brown-red amino compound - 3-amino-5-

constant weight. Accurately weigh 1.000g of glucose, dissolve it in distilled water and dilute it nitrosalicylic acid. Within a certain range, the amount of reducing sugar is directly proportional to the color

to 1000ml. Perform configuration operations according to Table 1, mix thoroughly, then heat of the reaction solution. In the literature [3-5], the measurement wavelengths of the DNS method are

and boil in a boiling water bath for 5 minutes. After cooling under running water, add 4ml of different, such as 540, 520, 490nm, etc. In order to further determine the optimal wavelength of the DNS

distilled water to each test tube and mix well. Using tube 1 as a blank control, measure the method in the process of measuring reducing power, spectrum scanning was performed considering

absorbance of each tube at a wavelength of 540nm. Draw an absorbance-glucose concentration curve. relevant factors. The results are as shown in the figure 1 and 2 shown.

Table 1 Preparation of glucose standard solution

2
Glucose chromogenic solution - water
Table 1 Preparation of standard glucose solution
1.5
Developer - water
Pipe number 123 4 56
Absorbance

1
Glucose chromogenic solution- chromogenic reagent
1mg/ml glucose solution (ml) 0 distilled 0.1 0.2 0.3 0.4 0.5
0.5
water (ml) 0.5 0.4 0.3 0.2 0.1 0
1.5 1.5 1.5 1.5 1.5 1.5 0
DNS reagent (ml)
460 480 500 520 540 560 580 600

Wavelength(nm)

1.3.3 Prepare acetic acid-sodium acetate buffer solution. Wavelength Scan for DNS Reagent 1

Solution A: 0.2mol/L acetic acid solution; Solution B: 0.2mol/L sodium acetate. Figure 1 Fig.1 Wavelength scanning of reagent one

solution. Prepare buffers with different pH (4.0~5.0) in proportion.

1.3.4 Determination of wavelength measured by DNS method Glucose chromogenic solution - water

2
Take 0.5ml glucose standard solution and 0.5ml distilled water and place them in 10ml test Developer - water
1.5
tubes respectively, then add 1.5ml DNS reagent each, heat in a boiling water bath for 5 minutes, cool Glucose chromogenic solution- chromogenic reagent
Absorbance

1
with running water, and adjust to volume with distilled water. Combine glucose color solution - water (blank),
0.5
DNS reagent-water (blank) and glucose chromogenic solution-DNS reagent (blank) were
0
scanned in the wavelength range of 400 to 600nm respectively. 460 480 500 520 540 560 580 600

Wavelength(nm)
1.3.5 Determination of DNS reagent dosage and storage time
Figure 2 Wavelength scanning of
Add 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0ml of DNS reagent to a series of 10ml test tubes in
DNS reagent 2Fig.2 Wavelength scanning of reagent two
which 0.5ml of glucose standard solution is accurately added , heat in a boiling water bath for

5 minutes, cool with running water, and set to volume. Use the same conditions without adding
During the experiment, the added amounts of DNS reagents in the blank and the solution to be
glucose standard solution as a blank solution, and measure the absorbance at different times
tested were the same before color development. However, during the color development, some of the DNS
at a wavelength of 540 nm.
reagents were combined with reducing sugars in the solution to be tested, which caused the DNS reagent in

1.3.6 Determination of heating time: Take

the blank to be measured when the absorbance was measured. The reagent content is greater than the DNS

0.5ml of glucose standard solution and 1ml of distilled water in a series of 10ml test tubes
reagent content in the liquid to be tested. It can be seen from Figure 1 that the wavelength at the maximum

, add DNS reagent respectively, place in boiling water bath to heat and react 1 ,
absorption peak is 500nm. In general, selecting the wavelength at the maximum absorption peak for

2, 3, 4, 5, 6, 7, 1 0, 1 5, 2 0, 2 5, 30min
measurement can improve sensitivity, but as shown in Figure 1, the DNS reagent also has a certain absorbance at this wavelength.

Take out, cool under running water, and set to volume. Use the same conditions without adding glucose
(DNS Reagent-Water Curve). Furthermore, during the experiment, the added amounts of DNS reagents in the

standard solution as a blank solution, and measure the absorbance of the solution at different heating times
blank and the solution to be tested were the same before color development. However, during the color

at a wavelength of 540 nm.

development, part of the DNS reagent combined with the reducing sugar in the solution to be tested, resulting in

1.3.7 For precision experiment , a blank when measuring the absorbance. The content of DNS reagent in the solution is greater than the content

take 10 tubes and add 0.5ml glucose standard solution and 1.5ml respectively. of DNS reagent in the solution to be tested. Therefore, at 500nm, DNS reagent seriously interferes with the measurement results.
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536 2008, Vol. 29, No. 08 food science ÿRiddle

Heavy. Combined with the glucose chromogenic solution-water curve, it can be seen that this effect is The added amount can be 1.5ml.

It is always significant for ÿ < 540 nm. Therefore, DNS reagent 1 in this experiment 2.4 Precision experiment

The final measurement wavelength was selected as 540nm instead of 500nm (maximum absorption It can be seen from Table 4 and Table 5 that under the conditions of this experiment, the performance of DNS reagent 1

wavelength). In the same way, it can be seen from Figure 2 that DNS reagent 2 is finally determined Precision is better than DNS reagent 2.

The measurement wavelength is 550nm, not 500nm (maximum absorption wavelength).

Table 2 Reagent 1 Precision experiment

2.2 Effect of heating time on measurement results Table 2 Precision experiment of reagent one

It can be seen from Figure 3 that as the heating time of the boiling water bath increases, the light absorption Tube No. 1 2 3 4 5 6 7 8 9 10

The absorbance becomes larger, but the absorbance of both reagents basically remains unchanged after 5 minutes. Absorbance 0.368 0.366 0.362 0.353 0.355 0.354 0.3600.3660.3630.734

Mean value 0.368 Standard error 0.001745 Standard deviation 0.005519

The obvious color reaction is basically completed and the absorbance is relatively stable. Therefore DNS reagent water

The bath heating time is 5 minutes.

Table 3 Precision experiment of reagent 2

Table 3 Precision experiment of reagent two

Reagent 1 Reagent 2
Tube No. 1 2 3 4 5 6 7 8 9 10
0.8
Absorbance 0.734 0.715 0.730 0.729 0.734 0.734 0.7240.7380.7170.734
0.6
Absorbance

Mean value 0.731 Standard error 0.003587 Standard deviation 0.011343

0.4

0.2 2.5 Standard curve

0 The standard curves of glucose for the two reagents are shown in Figure 6 . Standard
0 5 10 15 20 25
The line shows that the glucose concentration within the measurement range is sensitive, stable and
Time(min)

Figure Effect of DNS reagent heating time on measurement results into good linearity.

3 Figure 3 Effects of heating time on absorbance

1
Reagent 2 y=1.7329x - 0.0017
2.3 Determination of DNS reagent dosage and storage time
0.8 R2 =0.9992

0.6 Reagent 1
Absorbance

0.5ml 0.4
0.4 1.0ml y=0.7809x+0.0023
0.2
0.38 1.5ml R2 =0.9993
0
Absorbance

2.0ml 0 0.1 0.2 0.3 0.4 0.5 0.6

0.36
2.5ml
Glucose concentration (mg/ml)
0.34
3.0ml
0.32 Figure 6 DNS Reagent Glucose Standard Curve

0 50 100 150 Fig.6 Standard curve of two kinds of DNS reagents

Placement time(min)

Figure 4 Effect of reagent 1 placement time and dosage on absorbance 3 Conclusion

Fig.4 Effects of standing time and reagent one volume on

absorbance
In alkaline solution, 3,5-dinitrosalicylic acid is reduced by reducing sugar to produce

Amino compounds have maximum absorption at a wavelength of 500nm. But in this wave
0.8 0.5ml
1.0ml Long-term DNS reagent has a certain absorbance, which seriously interferes with the measurement results.
0.7 1.5ml
Absorbance

Heavy, so the best conditions for determining reducing sugar content by DNS method are DNS reagents.
2.0ml
0.6 2.5ml
Measure 1.5ml, keep in boiling water bath for 5min, dilute to volume and leave for 20min before measuring.
3.0ml
0.5 The detection wavelength of reagent 1 is 540nm, and the detection wavelength of reagent 2 is 550nm.
0 20 40 60 80 100

Placement time(min)
references:
Figure 5 Effect of reagent 2 placement time and dosage on absorbance
[1] Ning Zhengxiang. Food Composition Analysis Manual[M]. Beijing: China Light Industry Press,
Fig.5 Effects of standing time and reagent two volumes on
1998.
absorbance
[2] Commercial Standard of the People’s Republic of China SB/T10077-92 Technical Specifications for Noodle Production Process

[S].
It can be seen from Figures 4 and 5 that the reagent dosage, placement time and absorbance value
[3] Zheng Yuanbin, Wu Jinzhong. Determination of various sugar contents in lotus seeds by DNS colorimetric method[J]. Fujian Zhong

relationship, when the dosage of DNS reagent is less than 1.5ml, the absorbance of the solution changes with Journal of Medical College, 2004,14(4): 32-35.

[4] Yang Guiming, Jiang Aihua, Xue Qiusheng. Study on the conditions for determination of reducing sugars by DNS photometry[J].
The DNS reagent dosage increases with the increase. When the reagent dosage is 1.5~2ml,

Anhui Agricultural Sciences, 2006,34(14): 3258-3264.

The absorbance value is relatively stable after being left for 20 minutes. The dosage of reagent one is 2~3ml
[5] Sun Weiwei, Cao Weiqiang, Wang Jing. Determination of total sugar in corn straw by DNS method[J]. Food Research and

The absorbance value fluctuates greatly. In actual measurement, the two reagents
Development, 2006(6): 120-124.