Some standard content:
ICS 67.040
Baojia Standard of the People's Republic of China
GB/T 5009.85—2003
Replaces GB/T12391—1990
Determination of riboflavin in foods
Determination of riboflavin in foodsPublished on 2003-08-11
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on 2004-01-01
GB/T5009.85—2003
The first method of this standard corresponds to AOAC45.108 "Determination of riboflavin in foods and vitamin products by fluorescence" (1995 edition). The consistency degree of this standard with AOAC45.1.08 is non-equivalent. This standard replaces GB/T12391-1990 "Determination of riboflavin in food". Compared with GB/T12391-1990, the main changes of this standard are as follows: the Chinese name of the standard is changed to "Determination of riboflavin in food"; - The structure of the original standard is modified according to GB/T20001.4-2001 "Standard Preparation Rules Part 4: Chemical Analysis Methods".
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting unit of this standard: Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine. The main drafters of this standard: Wang Guangya, Qu Ning, Li Jing, Zhou Ruihua, Wang Zhu. The original standard was first issued in 1990, and this is the first revision. 616
1 Scope
Determination of riboflavin in food
This standard specifies the determination method of riboflavin content in food - microbiological method and fluorescence method. This standard is applicable to the determination of riboflavin in various foods. The detection limit of this method is 0.006μg for the fluorescence method; the linear range is 0.1μg~20μg for the fluorescence method. Method 1 Fluorescence method
2 Principle
GB/T 5009.85—2003
Riboflavin produces yellow-green fluorescence under 440nm~500nm wavelength light. In a dilute solution, its fluorescence intensity is proportional to the concentration of riboflavin. Its fluorescence intensity is measured at a wavelength of 525nm. Sodium hyposulfite (NazSzO.) is added to the test solution to reduce riboflavin to a non-fluorescent substance, and then the fluorescence intensity of the residual fluorescent impurities in the test solution is measured. The difference between the two is the fluorescence intensity generated by riboflavin in food.
3 ReagentsbzxZ.net
3.1 Silicon magnesium adsorbent: 60 mesh~100 mesh. 3.22.5mol/L sodium acetate solution.
3.3 Papain (100g/I.): Prepare with 2.5mol/L sodium acetate solution. Prepare immediately when using. 3.4 Amylase (100g/L): Prepare with 2.5mol/L sodium acetate solution. Prepare immediately when using. 3.50.1mol/L hydrochloric acid.
3.61mol/L sodium hydroxide.
3.70.1mol/L sodium hydroxide.
3.8 Sodium hyposulfite solution (200g/1): Prepare immediately when using. Store in an ice water bath and it is valid within 4 hours. Eluent: acetone + glacial acetic acid decahydrate (5+2+9). 3.9
3.10 Bromocresol green indicator (0.4g/L). 3.11 Potassium permanganate solution (30g/L).
3.12 Hydrogen peroxide solution (3%).
3.13 Preparation of riboflavin standard solution (purity 98%) 3.13.1 Riboflavin standard stock solution (25ug/mL): Place the standard riboflavin powder crystals in a vacuum dryer or a dryer containing sulfuric acid. After 24 hours, accurately weigh 50mg, place it in a 2I volumetric flask, add 2.4mL glacial acetic acid and 1.5L water. Shake the volumetric flask in warm water until it dissolves, cool to room temperature, dilute to 2L, transfer to a brown bottle, add a little toluene to cover the surface of the solution, and store in a refrigerator.
3.13.2 Riboflavin standard working solution: Take 2.00mL of riboflavin standard stock solution, place it in a 50m. brown volumetric flask, and dilute to the mark with water. Protect from light and store in a 4℃ refrigerator for one week. This solution is equivalent to 1.00μg riboflavin per milliliter. 4 Instruments
4.1 Common laboratory equipment
4.2 High pressure sterilizer.
4.3 Electric constant temperature incubator.
GB/T5009.85-2003
4.4 Riboflavin adsorption column: see Figure 1.
4.5 Fluorescence spectrophotometer.
5 Analysis steps
The whole operation process should be carried out in the dark.
5.1 Sample extraction
5.1.1 Sample hydrolysis
Figure 1 Riboflavin adsorption column
Accurately weigh 2g~~10g sample (containing about 10g~200g riboflavin) in a 100mL conical flask, add 50mL 0.1mol/L hydrochloric acid, and stir until the particles are evenly dispersed. Cover the bottle mouth with a 40mL porcelain crucible, place it in a high pressure cooker for high pressure hydrolysis, 10.3×10*Pa30min. After the hydrolyzate is cooled, add 1mol/L sodium hydroxide dropwise, take a small amount of the hydrolyzate, and test it with 0.4g/L bromocresol green to find that it is grass green, with a pH of 4.5.
5.1.2 Enzymatic hydrolysis of the sample
5.1.2.1 Hydrolyzate containing starch: add 3mL 10g/L amylase solution, and keep warm at 37℃~40℃ for about 16h. 5.1.2.2 Hydrolyzate containing high protein: add 3mL 10g/L papain solution, and keep warm at 37℃~40℃ for about 16h. 5.1.3 Filtration
The above enzymatic hydrolyzate is diluted to 100.0mL and filtered with dry filter paper. This extract can be stored in a refrigerator at 4℃ for one week. 5.2 Oxidation to remove impurities
Depending on the riboflavin content in the sample, take a certain volume of sample extract and riboflavin standard solution (containing about 1μg to 10ug riboflavin) in a 20mL capped graduated test tube and add water to 15mL. Add 0.5mL of glacial acetic acid to each tube and mix well. Add 0.5mL of 30g/L potassium permanganate solution, mix well, and let stand for 2min to oxidize and remove impurities. Add a few drops of 3% hydrogen peroxide solution until the color of potassium permanganate fades. Shake the tube vigorously to allow excess oxygen to escape. 5.3 Adsorption and elution of riboflavin
5.3.1 Riboflavin adsorption column: About 1g of silicon magnesium adsorbent is loaded into the column by wet method, and it is appropriate to occupy 1/2~~2/3 of the column length (about 5cm) (a small ball of absorbent cotton pad is used to pad the lower end of the adsorption column). Do not allow bubbles to form in the column, and adjust the flow rate to about 60 drops/min. 5.3.2 Column and elution: After all the oxidized sample solution and standard solution have passed through the adsorption column, use about 20 mL of hot water to wash away the impurities in the sample solution. Then use 5.00 mL of eluent to elute the riboflavin in the sample and collect it in a 10 mL graduated test tube with a cap. Then wash the adsorption column with water, collect the washed liquid and make it up to 10 mL, mix it and then measure the fluorescence. 618
5.4 Preparation of standard curve
GB/T5009.85—2003
Accurately pipette 0.30.6, 0.9, 1.25, 2.5, 5.0, 10.0, 20.0mL of riboflavin standard solution (equivalent to 0.3, 0.6, 0.9, 1.25, 2.5, 5.0, 10.0, 20.0μg riboflavin) respectively, or take a single point standard with a content close to that of the sample and follow the steps of riboflavin adsorption and elution (5.3).
5.5 Determination
5.5.1 Measure the fluorescence value of the sample tube and the standard tube at an excitation wavelength of 440nm and an emission wavelength of 525nm. 5.5.2 After the fluorescence value of the sample and standard is measured, add 0.1mL20% sodium hyposulfite solution, mix immediately, measure the fluorescence value of each tube within 20s, and make the blank value of each tube. 6 Calculation of results
See formula (1).
Wherein:
× - (2=B×+×100
(C--D) X m
X—-Riboflavin content in the sample, in milligrams per hundred grams (mg/100g); A——Fluorescence value of the sample tube;
B-——Blank fluorescence value of the sample tube;
-Fluorescence value of the standard tube;
Blank fluorescence value of the standard tube:
Dilution multiple;
Sample mass, in grams (g);
-Riboflavin mass in the standard tube, in micrograms (ug); 100
The riboflavin in the sample The coefficient for converting the content from micrograms per gram (μg/g) to milligrams per hundred grams (mg/100g). The calculated result is expressed to two decimal places. 7 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. Second method Microbiological method
8 Principle
(1)
Certain vitamins are necessary for the growth (reproduction) of a certain microorganism. For example, Lactobacillus casei (Lactobacillus casei, referred to as LC) requires riboflavin for growth. If the culture medium lacks this vitamin, the bacteria cannot grow. Under certain conditions, the growth of the bacteria and the concentration of its metabolite lactic acid are proportional to the content of the vitamin in the culture medium. Therefore, the acidity and turbidity determination method can be used to determine the riboflavin content in the sample.
9 Reagents
9.1 Glacial acetic acid.
9.2 Toluene.
9.3 Anhydrous sodium acetate.
9.4 Lead acetate.
9.5 Ammonium hydroxide.
GB/T5009.85--2003
9.6 Lactobacillus casei ATCC 7469) 9.7 Hydrochloric acid: 0.1 mol/L.
9.8 Sodium hydroxide solution: 1 mol/L and 0.1 mol/L. 9.9 0.9 g/L sodium chloride solution (normal saline): sterilize before use. 9.10 Riboflavin standard stock solution (25 μg/mL): place the standard riboflavin powder crystals in a vacuum dryer or a dryer filled with sulfuric acid. After 24 hours, accurately weigh 50 mg, place in a 2L volumetric flask, add 2.4 mL glacial acetic acid and 1.5 L water. Shake the volumetric flask in warm water until it dissolves, and cool to room temperature. Warm, dilute to 2L, transfer to a brown bottle, add a little toluene to cover the surface of the solution, and store in a refrigerator.
9.11 Riboflavin standard intermediate solution (10ug/mL): Accurately pipette 20mL of riboflavin standard stock solution, add water to dilute to 50mL. 9.12 Riboflavin standard working solution (0.1μg/mL): Accurately pipette 1.0mL of intermediate solution into a 100mL volumetric flask, add water to dilute to the scale, and shake well. Prepare a new standard working solution for each analysis. 9.13 Alkali-treated protein chen: Weigh 40g of protein chen and 20g of sodium hydroxide in 250mL of water. After mixing, place in a constant temperature box at 37℃±0.5℃, and take out after 24h~48h. Use glacial acetic acid to adjust pH to 6.8, add 14g of anhydrous sodium acetate (or 23.2g of sodium acetate containing 3 molecules of crystal water), dilute to 800mL, add a little toluene to cover the surface of the solution, and store in a refrigerator. 9.14 Cystine solution (1g/L): Weigh 1g L-cystine in a small beaker. Add 20mL of water, slowly add about 5mL to 10mL of hydrochloric acid until it is completely dissolved, dilute to 1I with water, and add a little toluene to cover the surface of the solution. 9.15 Yeast supplement solution: Weigh 100g of yeast extract powder in 500mL of water, weigh 150g of lead acetate in 500mL of water, mix the two solutions, and adjust the pH with ammonium hydroxide until phenolphthalein turns red (take a little solution for inspection). Centrifuge or filter with a Buchner funnel, and adjust the pH of the filtrate to 6.5 with glacial acetic acid. Pass hydrogen sulfide until no precipitation occurs, filter, and pass air into the filtrate to remove excess hydrogen sulfide. Add a little toluene to cover the surface of the solution and store in a refrigerator. 9.16 Methyl salt solution: Weigh 25g of dipotassium hydrogen phosphate and 25g of potassium dihydrogen phosphate, dissolve in water, and dilute to 500mL. Add a little toluene to preserve it.
9.17 Ethyl salt solution: Weigh 10g magnesium sulfate (MgSO4·7H2O), 0.5g ferrous sulfate (FeSO4·7H2O) and 0.5g manganese sulfate (MnSO4·4H,O) and dissolve them in water, dilute to 500mL, and add a little toluene to preserve it. 9.18 Basic culture stock solution: Mix the following reagents in a 500mL beaker, add water to 450mL, adjust the pH to 6.8 with 1mol/L sodium hydroxide solution, and dilute to 500mL with water. Alkali-treated protein Chen
0.1% cystine solution
Yeast supplement
A salt solution
B salt solution
Anhydrous glucose
100 mL
9.19 Agar medium: Mix the following reagents in a 250mL Erlenmeyer flask, add water to 100mL, boil in a water bath until the agar is completely dissolved, and adjust the pH to 6.8 with 1mol/L hydrochloric acid while hot. Pour into test tubes as soon as possible, 3mL~5mL per tube, plug with cotton, sterilize in an autoclave at 6.9×104 Pa pressure for 15 min, take out and stand the test tube upright, cool to room temperature, and store in a refrigerator. Anhydrous glucose
Sodium acetate (NaAc·3H,0)
Protein Chen
Yeast extract in powder
Methyl salt solution
Ethyl salt solution
9.200.4g/L Bromocresol green indicator: weigh 0.1g bromocresol green in a small mortar, add 1.4mL0.1mol/1.620 sodium hydroxide solution
grind. Add a little water and continue to grind until completely dissolved. Dilute with water to 250ml. GB/T5009.85—2003
9.210.4g/L Bromothymol blue indicator: weigh 0.1g bromothymol blue in a small mortar, add 1.6mL0.1mol/L sodium hydroxide solution and grind. Add a little water and continue to grind until completely dissolved. Dilute with water to 250mL. 10 Instruments and equipment
10.1 Common laboratory equipment.
10.2 Electric constant temperature incubator.
10.3 Centrifugal sedimentation machine.
10.4 Liquid rapid mixer.
10.5 High pressure sterilizer.
11 Preparation and preservation of bacterial strains
11.1 Preparation of reserve bacterial strains: Inoculate LC pure bacterial strains into 2 or more agar culture tubes. Keep warm in a constant temperature incubator at 37℃±0.5℃ for 16h~24h. Store in a refrigerator for no more than 2 weeks, and it is best to transplant once a week. Reserve bacterial strains that have been stored for more than a few weeks cannot be used immediately to prepare inoculation liquid. They must be transplanted once a day before use for 2d~3d in a row before use, otherwise they will not grow well. 11.2 Preparation of seed culture solution: Take 5ml of riboflavin standard solution and 5ml of basic culture reserve solution in a 15ml centrifuge tube, mix well, plug with cotton, and sterilize in a pressure cooker at 6.9×104Pa for 15min. 2 to 4 tubes can be prepared each time. 12 Analysis steps
Since riboflavin is easily destroyed by sunlight and ultraviolet rays, all operations should be carried out in a dark room. 12.1 Preparation of inoculation solution
The day before use, transfer the strain from the reserve strain tube into the sterilized seed culture solution, and prepare two tubes at the same time. Keep warm at 37℃±0.5℃ for 16h~24h. After taking out, centrifuge for 10min (3000r/min), pour off the upper liquid in a sterile operation method, rinse twice with sterilized physiological saline, add 10ml of sterilized physiological saline, shake the test tube on a liquid rapid mixer to make the strain into a suspension. Pour this liquid into a sterilized syringe and use it immediately. 12.2 Preparation of the sample
12.2.1 Grind into powder with a grinder or mortar or beat into a homogenate with a crusher. 12.2.2 Weigh a sample containing about 5μg to 10uμg of riboflavin (about 10g for cereals, about 4g for beans, and about 5g for meat), add 50ml of 0.1mol/L hydrochloric acid solution, and mix well. Place in a pressure cooker and hydrolyze at a pressure of 10.3×104Pa for 30min. 12.2.3 Cool to room temperature and adjust the pH to 4.6 with 1mol/L sodium hydroxide solution (take a small amount of the hydrolyzate and test it with bromocresol green. The solution is grass green).
12.2.4 Add amylase or papain, adding 20mg of enzyme per gram of sample. Place in a 40℃ constant temperature box overnight, about 16h. 12.2.5 Cool to room temperature and dilute to 100mL with water.Filter. For foods with high fat content, ether can be used for extraction to remove fat. 12.3 Preparation of standard tubes
Add 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mL of riboflavin standard solution to each of the two test tubes, add water to each tube to make it 5 mL, and then add 5 mL of basic culture stock solution to each tube and mix well. 12.4 Preparation of sample tubes
12.4.1 Take 5 mL to 10 mL of the sample solution and place it in a 25 mL stoppered test tube, adjust the pH to 6.8 with 0.1 mol/L sodium hydroxide (take a small amount of the solution and test it with bromovanillin blue), and dilute it to the mark with water. 12.4.2 Take two test tubes and add 1, 2, 3, 4 mL of the sample diluent (12.4.1) to each tube, add water to each tube to make it 5 mL, and then add 5 mL of basic culture stock solution to each tube and mix well.
12.5 Sterilization
Plug all the above sample tubes and standard tubes with cotton plugs, place them in a pressure cooker, and sterilize them at a pressure of 6.9×10°Pa for 15 minutes. 621
GB/T5009.85—2003
12.6 Inoculation and culture
12.6.1After the test tubes cool to room temperature, inoculate them under aseptic operation conditions, and add one drop of inoculation solution (12.1) to each tube. When inoculating, the syringe needle should not touch the wall of the test tube, and the inoculation solution should be directly dropped into the culture solution. 12.6.2 Place in a 37℃±0.5℃ constant temperature box and culture for about 72 hours. During the culture, each tube must be at the same temperature. The culture time can be extended by 18 hours or reduced by 12 hours. If necessary, it can be stored in a refrigerator overnight and then titrated. If the turbidity determination method is used, it is appropriate to culture for 18 hours to 24 hours. 12.7 Titration
Pour the culture solution in the test tube into a 50mL conical flask, add 5mL of 0.01g/L bromothymol blue solution, rinse the test tube twice, pour the washing solution into the conical flask, and titrate with 0.1mol/L sodium hydroxide solution. The end point is green. Use the titration end point of the first bottle as the color change reference bottle. After about 30 minutes, change to another reference bottle because the color of the solution becomes lighter due to being left for too long. 12.8 Drawing of standard curve
Draw a standard curve using different concentrations of standard riboflavin solution as the horizontal axis and the number of milliliters of 0.1mol/L sodium hydroxide required during titration as the vertical axis.
13 Calculation of results
See formula (2).
Wherein:
X=x×F×10%
X-riboflavin content in the sample, in milligrams per hundred grams (mg/100g); riboflavin content in each milliliter of the sample is obtained by the curve, in micrograms per milliliter (μg/mL); V
total volume of the sample hydrolyzate, in milliliters (mL); F
dilution factor of the sample solution;
mass of the sample, in grams (g);
the coefficient calculation result of converting the sample content from micrograms per gram (μg/g) to milligrams per hundred grams (mg/100g) is expressed to the second decimal place. 14 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 622
·（2)
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