GB/T 14924.11-2001 Determination of vitamins in compound feeds for experimental animals
Some standard content:
GB/T14924.11—2001
All technical contents of this standard are recommended. It was separated from the previous "Complete Nutrient Feed" to form an independent standard. This standard is equivalent to the two methods listed in GB14924--1994 "Laboratory Animals". This standard and its supporting standards shall replace GB14924-1994 from the date of implementation. This standard is proposed and managed by the Ministry of Science and Technology of the People's Republic of China. Drafting unit of this standard: Chinese Society of Laboratory Animals The main drafters of this standard: Zhou Ruihua, Wang Zhu, Shi Lei, Wang Guangya, Zhang Yu, Zheng Tao, Liu Xiumei. This standard is entrusted by the Ministry of Science and Technology of the People's Republic of China to the Chinese Society of Laboratory Animals for technical coordination to interpret this standard. This standard was first issued in January 1994.
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1 Scope
National Standard of the People's Republic of China
Laboratory animals-Formula feeds-Determination of vitamins
Laboratory animals-Formula feeds-Determination of vitamins
GB/T 14924.11--2001
Replaces GB149241994
This standard specifies the determination method of vitamins in laboratory animal formula feeds, namely, the determination method of vitamin A, vitamin E, vitamin B, vitamin B2, niacin, vitamin B6, total ascorbic acid, total choline, folic acid, vitamin B12, vitamin K3, pantothenic acid, biotin, vitamin D in formula feeds.
This standard is applicable to the determination of formula feeds and their raw materials for laboratory animals such as mice, rats, rabbits, guinea pigs, hamsters, dogs and monkeys. 2 Reference Standards
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard is published, the versions shown are valid. All standards will be revised, and the parties using this standard should explore the possibility of using the latest versions of the following standards. GB/T 12388—1990 Method for determination of vitamin A and vitamin E in foods GB/T 12390-1990
GB/T 12391---1990
GB/T 12392—1990
GB/T 12395—1990
GB/T 14700--1993
GB/T 14701—1993
GB/T 17407-
GB/T 17812—1999
GB/T 17816—1999
GB/T 17817—1999
GB/T 17818--1999
3 Determination methods
Determination method of thiamine (vitamin B12) in foodDetermination method of riboflavin in food
Determination method of total ascorbic acid in vegetables, fruits and their productsFluorescence method and 2,4-dinitroaniline methodDetermination method of niacin in food
Determination method of vitamin B12 in feed
Determination method of vitamin B2 in feed
Determination of vitamin B2 in food
Determination of vitamin E in feedHigh performance liquid chromatographyDetermination of total ascorbic acid in feedO-phenylenediamine fluorescence methodDetermination of vitamin A in feedHigh performance liquid chromatographyDetermination of vitamin D in feedHigh performance liquid chromatography3.1 The determination of vitamin A and vitamin E in compound feed shall be carried out in accordance with the provisions of GB/T12388, GB/T17812 and GB/T17817. 3.2 Determination of vitamin B1 in compound feeds
shall be carried out in accordance with the provisions of GB/T14700 and GB/T12390. 3.3 Determination of vitamin B2 in compound feeds
shall be carried out in accordance with the provisions of GB/T12391 and GB/T14701. Approved by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China on August 29, 2001
Implemented on May 1, 2002
3.4 Determination of niacin in compound feeds
shall be carried out in accordance with the provisions of GB/T12395.
3.5 Determination of vitamin B2 in compound feeds
shall be carried out in accordance with the provisions of GB/T17407.
3.6 Determination of total ascorbic acid in compound feed GB/T 14924.11
According to GB/T12392 and GB/T17816. 3.7 Determination of total choline in compound feed
3.7.1 Principle
Choline in compound feed is extracted by alkali treatment, purified by silica-magnesium adsorbent column chromatography, and then reacted with reineckate to form a pink choline-reineckate complex. After the complex is eluted with acetone, it has a maximum absorption at 526nm, and its absorption value is proportional to the concentration of reineckate. The detection limit of this method is 0.1mg. 3.7.2 Reagents
All reagents are analytical grade, and the experimental water is distilled water. 3.7.2.1 Methanol.
3.7.2.2 Chloroform.
Methyl acetate.
3.7.2.4 Radone.
3.7.2.5 10% acetone: Mix 10 ml of acetone with 90 ml of water. 3.7.2.6
Glacial acetic acid.
Glacial acetic acid-methanol solution: Mix 10 ml of glacial acetic acid with 90 ml of methanol. 3.7.2.8
Barium hydroxide.
3.7.2.9 Silicon magnesium adsorbent (Florisil): 60~100 mesh. 3.7.2.10 Extraction solution: Add 4~5g of anhydrous barium hydroxide to 100 ml of methanol, stir for 10 minutes, then add 10 ml of methyl chloride and mix, filter to remove excess barium hydroxide. 3.7.2.11 Saturated solution of ammonium reineckate: weigh 2-3 g of ammonium reineckate, add 100 ml of water, stir for 10 min, and filter to remove excess ammonium reineckate. Prepare on the day of the experiment. 3.7.2.12 Choline standard stock solution (5 mg/mL): accurately weigh 0.5761 g of anhydrous choline chloride, dissolve in water, and dilute to 100 ml. Store in a refrigerator.
3.7.2.13 Choline standard application solution (1.0 mg/mL): accurately aspirate 20.0 ml of standard stock solution, dilute with water and dilute to 100 ml. 3.7.3 Instruments and equipment
3.7.3.1 Common laboratory equipment.
3.7.3.2 Reflux extraction device.
3.7.3.3 Chromatographic column: 0.8cm (inner diameter) × 30cm glass column, with a liquid storage cup of 30-50mL at the top of the column, and a piston at the bottom. There is a glass sieve plate about 1cm above the piston, and the pore size of the sieve plate is 16-30μm. It needs to be dried before use. 3.7.3.4 Spectrophotometer.
3.7.4 Determination steps
3.7.4.1 Extraction
Weigh an appropriate amount of sample (containing about 5-50mg choline), place it in a 100ml stoppered conical flask, add 40mL of extract, and reflux in a constant temperature water bath at 76-82℃ for 3h, with a reflux rate of 1-2 drops per second. Cool, filter the sample into a 100mL volumetric flask, repeatedly wash the conical flask and the filter residue with 5-10mL glacial acetic acid-methanol solution, add the washing liquid into the volumetric flask, adjust the pH to 2-6 with glacial acetic acid, and dilute to the mark with methanol.
3.7.4.2 Purification
3.7.4.2.1 Filling the chromatographic column: Soak about 4g of dry silica-magnesium adsorbent in methanol, take it from the dry chromatographic column, and fill the silica-magnesium adsorbent into the chromatographic column by wet method until the height of the silica-magnesium adsorbent reaches about 10cm. Rinse the chromatographic column with methanol and keep the methanol liquid level 1-2cm higher than the silica-magnesium adsorbent until use.
3.7.4.2.2 Column chromatography purification: Pipette 50 ml of the extract and add it to the packed column. Open the piston at the bottom and let the extract flow through the column by gravity. Immediately wash the column with 5, 10 mL of methanol, 2 10 mL of methyl acetate and 10 mL of 10% acetone. Then add 5 mL of saturated Reinacke's ammonium salt solution through the column and wash with 2 10 mL of glacial acetic acid until the effluent is clear. Elute the pink chromatographic band of the choline-Reinacke's salt complex on the column with 15 ml of acetone. Collect all the eluate with a 25 mL stoppered measuring cylinder and dilute to 15 mL with acetone.
3.7.4.3 Colorimetric determination: Use a spectrophotometer at a wavelength of 526nm, adjust the zero point with raditin, determine the sample absorbance value, find the choline content on the standard working curve, or use the regression equation to calculate the corresponding choline content and obtain the calculation result. 3.7.4.4 Standard working curve: Take 0.50, 1.0, 2.0, 3.0, 4.0, 5.0mL of standard application solution, which is equivalent to 0.5, 1.0, 2.0, 3.0, 4.0, 5.0mg of choline content, respectively, and follow the above sample determination steps. Use the choline content as the horizontal axis and the absorbance value as the vertical axis to draw the standard working curve, and calculate the regression equation. 3.7.5 Calculation result
See formula (1).
m××100
Where: X.--Choline content in the sample, mg/100gc--Choline content obtained from the standard regression curve, mg; V,--Volume of sample extract, mL, V2\---Volume of extract for purification, mL; mSample mass, g.
3.7.6 Permissible difference of results
The absolute value of the relative deviation of the results of parallel or repeated determinations in the same laboratory is ≤10%. 3.8 Determination of folic acid in compound feed--microbiological method 3.8.1 Principle
Folic acid is a nutrient necessary for the growth of Lactobacillus casei (LC, ATCC7469). Under certain conditions, the growth and reproduction of IC is proportional to the folic acid content in the culture medium, and the bacterial proliferation intensity is expressed by the measured absorbance value. Compare with the standard curve to calculate the folic acid content in the sample. The detection limit of this method is 0.1ng. 3.8.2 Reagents
All water used in this experiment is distilled water. The purity of reagents is analytical grade unless otherwise specified. 3.8.2.1 Toluene.
3.8.2.2 Physiological saline: Sterilize before use. 3.8.2.3 Bacterial strain: Lactobacillus casei (LC, ATCC7469) 3.8.2.4 Phosphate buffer (0.05 mol/L, pH 6.8): Weigh 4.35 g of sodium phosphate (NasPO.12H,0) and 10.39 g of disodium hydrogen phosphate (Naz2HPO.·7HzO) and dissolve them in 800 mL of water. Before use, add about 5g ascorbic acid and adjust the pH to 6.8. 3.8.2.5 Chicken pancreatin: weigh 100mg dried chicken pancreatin, add 20mL phosphate buffer to make a homogenate, centrifuge at 3000r/min for 10min, take the supernatant for use, and prepare before use. 3.8.2.6 Protease-amylase: weigh 200mg of protease and amylase respectively, add 20ml phosphate buffer to make a homogenate, centrifuge at 3000r/min for 10min, take the supernatant for use. Prepare before use. 3.8.2.7 Sodium hydroxide solution (0.01mol/L): Prepare with 20% ethanol. 3.8.2.8 Sodium hydroxide solution (10mol/L). 3.8.2.9 Folic acid standard stock solution (200μg/ml): accurately weigh 200mg folic acid standard, dissolve with 0.01mol/l sodium hydroxide solution and dilute to 11. Store in a brown bottle. 17
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GB/T 14924.11 --2001
3.8.2.10 Folic acid standard intermediate solution (200ng/mL): Pipette 1.0mL folic acid standard stock solution, dilute to 11.0 with 0.01mol/. sodium hydroxide solution. Store in a brown bottle. 3.8.2.11 Folic acid standard application solution (0.2ng/mL): Pipette 1.0ml folic acid standard intermediate solution, dilute to 11.0 with phosphate buffer.
3.8.2.12 Enzymatic casein: Dissolve 8g sodium bicarbonate in 1L water, add 60g vitamin-free casein, and adjust pH to 8.0 with 10mol/L sodium hydroxide solution. Add 300mg pancreatin and stir for 20min to fully mix the pancreatin. Add 2.5 ml of toluene and place in a 37°C thermostat for enzymatic hydrolysis for 48 to 72 hours. Take the casein out of the thermostat and terminate the reaction by high pressure at 121°C for 30 minutes to remove the toluene. Cool, add 10 g of diatomaceous earth (technical grade) and stir, and filter with a Buchner funnel. Add about 60 mL of glacial acetic acid to the filtrate to adjust the pH to 3.7. Weigh 12 g of activated carbon, add it to the filtrate and stir for 10 minutes, filter with a Buchner funnel, and repeat three times. Each time you filter, add 10 g of diatomaceous earth to the Buchner funnel to assist in filtration. Finally, dilute the filtrate with water to 1200 mL and store in the refrigerator. Take a small portion of the enzymatic casein solution for drying. If the solid content is less than 40 mg/mL, it needs to be prepared again.
3.8.2.13 Xanthine solution: Take 0.4 g of xanthine, add 10 mL of ammonia water, heat to dissolve, and dilute with water to 100 mL. Store in the refrigerator. 3.8.2.14 Adenine-purine-uracil solution: Weigh 0.2g of adenine sulfate, guanine hydrochloride and uracil respectively, add (1+4) hydrochloric acid solution, heat to dissolve, dilute with water to 100ml and store at room temperature. 3.8.2.15 Acetate buffer (1.7mol/L, pH4.5): 38.65g sodium acetate, 19.8mL acetic acid, add water to 500mL. 3.8.2.16 Vitamin solution: Dissolve 10mg riboflavin in 40mL acetate buffer. Dissolve 0.2mg biotin, 2.5mg sodium bicarbonate, 20mg p-aminobenzoic acid, 40mg pyridoxine hydrochloride, 4mg thiamine hydrochloride, 8mg calcium pantothenate, and 8mg niacin in 50mL water. Mix the above two solutions and add water to 100ml. 3.8.2.17 Tween-80 solution: Add 2g Tween-80 to 100mL 45C water and mix. 3.8.2.18 Reduced glutathione solution: Take 0.1g reduced glutathione and dissolve it in water to 100mL. 3.8.2.19 A salt solution: Weigh 5g dipotassium hydrogen phosphate and 2g potassium dihydrogen phosphate, dissolve them in water to 100mL, and add a little toluene to the liquid surface to preserve it.
3.8.2.20 Ethyl salt solution: Weigh 2g magnesium sulfate, 0.5g ferrous sulfate and 0.5g manganese sulfate, dissolve them in water to 100mL, and add a little toluene to the liquid surface to preserve it.
3.8.2.21 Basic culture medium: 100mL of enzymatic casein, 2.5mL of adenine-guanine-uracil solution, 2.5mL of xanthine solution, 5mL of vitamin solution, 2.5mL of Tween-80 solution, 0.3g of L-aspartic acid, 0.2g of L-cysteine hydrochloride, 2.5ml of reduced glutathione solution, 20g of glucose, 20g of sodium acetate, 2.5mL of methyl salt solution, add water to 250mL, stir, adjust pH to 6.8±0.1 with sodium hydroxide solution, then add 2.5mL of methyl salt solution, 200mL of phosphate buffer, and make up to 500mL with water. It can be stored in the refrigerator for one week. 3.8.2.22 Agar medium: glucose 1g, protein 0.8g, yeast extract powder 0.2g, sodium acetate (NaAc·3H20) 1.7g, salt A solution 0.2mL, salt B solution 0.2ml, agar 1.2g, add water to 100mL, put in a water bath and cook until agar is completely melted, adjust pH to 6.8±0.1. Pour into test tubes as soon as possible, plug 3-5ml of each tube with cotton plugs, sterilize at 121C for 15min, take out and stand the test tube upright, cool to room temperature, and store in a refrigerator.
3.8.3 Instruments and equipment
3.8.3.1 Common laboratory equipment.
3.8.3.2 Constant temperature incubator.
3.8.3.3 Centrifuge.
3.8.3.4 High pressure sterilizer.
3.8.3.5 Oscillator.
3.8.3.6 Inoculation needle and inoculation loop.
3.8.3.7 Spectrophotometer.
3.8.4 Preparation and preservation of bacterial strains
3.8.4.1 Preparation of reserve bacterial strains: Transfer the LC pure bacterial strains to 2 or more agar culture tubes. Culture in a (37±0.5)°C constant temperature incubator for 16-24 hours. Store in a refrigerator and transfer the strains once a week for reserve bacterial strains. 518
GB/T14924.11 ----2001
3.8.4.2 Preparation of seed culture medium: Take 2mL of folic acid standard application solution and 10ml of basic culture medium, mix well, and dispense into 4 5ml centrifuge tubes, plug with cotton wool, sterilize at 121C for 15 min, and set aside. 3.8.5 Determination steps (all operations must be carried out in the dark) 3.8.5.1 Preparation of inoculum: The day before use, transfer the strain from the reserve strain tube to 2 sterilized seed culture medium, (37±0.5) (incubate in a constant temperature incubator for 16-24h. The next morning, suspend the seed culture medium, and aseptically pipette 0.2ml, transfer the bacteria to another 2 sterilized seed culture medium, and culture at (37±0.5)C for another 6h. Take out 3000r/min, centrifuge for 10min, pour off the supernatant, wash twice with sterilized saline, centrifuge, and remove the supernatant. Finally, add 5mL of saline, shake and mix, and make a bacterial culture solution. Use immediately.
3.8.5.2 Sample preparation
3.8.5.2.1 Sample treatment: Grind the sample into powder. 3.8.5.2.2 Hydrolysis: Weigh 0.1~~0.5g sample (containing about 100~300ng of folic acid) in a 100mL conical flask, add 50mL of phosphate buffer, and mix. 121 (high pressure hydrolysis for 15 min. 3.8.5.2.3 Enzymolysis: After the hydrolysis sample is cooled, add 1 ml chicken pancreatin, 1 ml protease-amylase, and 1 mL toluene, mix thoroughly (37+0.5) (enzymolysis in a constant temperature incubator for 1620 h. Take out the enzymolysis sample and dilute it to 100 mL with water, filter it. Take 2 mL of the filtrate and dilute it to 20 nml., so that the final folic acid content is within the range of 0.1~0.3 ng/ml. At the same time, take another test tube, add 1 ml chicken pancreatin, 1 ml protease-amylase, and Enzyme blank control.
3.8.5.3 Preparation of standard series tubes
Take 2 sets of parallel test tubes, add 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0mL of folic acid standard application solution to each tube, which is equivalent to 0.0, 1.0, 0.2, 0.4, 0.6, 0.8, 1.0ng of folic acid content, add water to 5.0ml, add 5mL of basic culture medium, and mix well. 3.8.5.4 Preparation of sample tubes
Take 4 sets of test tubes, add 1.0mL of enzymatic hydrolysis sample to each tube, .2.0, 3.0.4.0ml, add water to a volume of 5.0ml, then add 5ml of basic culture medium and mix.
3.8.5.5 Sterilization: Plug all the above standard series tubes, sample tubes and enzyme blank tubes with cotton plugs and sterilize them at 121℃ for 15min. 3.8.5.6 Inoculation: After the standard series tubes, sample tubes and enzyme blank tubes cool to room temperature, inoculate under sterile operating conditions, inoculate one drop of inoculation solution per tube, and drop it directly into the culture medium. Leave a standard ○ tube uninoculated for zeroing when measuring absorbance. 3.8.5.7 Cultivation: Place in a (37 ± 0.5) ℃ constant temperature incubator for 20~40h. 3.8.5.8 Determination: Use a spectrophotometer at a wavelength of 540nm and adjust the absorbance value of the uninoculated standard 0 tube to 0. Determine the absorbance values of the standard tube, sample tube and enzyme blank.
3.8.5.9 Draw a standard curve
Draw a standard curve with the folic acid standard series content as the horizontal axis and the absorbance value as the vertical axis. 3.8.6 Calculation of results
See formula (2).
X = (c- P)XVX10 × 100
m X 1 000
Where: X·-folic acid content in the sample, μg/100g; c---folic acid content in each milliliter of sample determination tube obtained from the standard curve, ng/mL; p. folic acid content in the enzyme blank control tube, ng /ml.;V…Total volume of sample enzymatic solution, mL;mSample mass.g;
10---Dilution factor;
100/1000--Coefficient for converting sample content from ng/g to μg/100g. 3.8.7 Allowable error
Inter-laboratory parallel determination or repeated determination results relative absolute value <10%. (2)
3.9 Determination of vitamin B in compound feed
3.9.1 Principle
GB /T14924.112001
Vitamin Br is an essential nutrient for the normal growth of Tuctobucillusleichmannii (ATCC7830). Under fixed growth conditions, the growth and reproduction of Luctobucillusleichmuannii is linearly related to the content of vitamin Bu in the solution. The intensity of bacterial growth and reproduction can be measured by absorbance measurement to calculate the content of vitamin B in food and feed samples. The minimum detection limit of this method is 0.001 ng.
3.9.2 Reagents
The water used in this test is distilled water, and all reagents used must be analytically pure reagents. 3.9.2.1 Toluene
3.9.2.2 Citric acid (C,H.0,·3H,0). 3.9.2.3 Sodium hydrogen phosphate (Na2HPO),). 3.9.2.4 Sodium metabisulfite (NazS.O,). 3.9.2.5 Ascorbic acid (biochemical reagent). 3.9.2.6 Anhydrous glucose.
3.9.2.7 Anhydrous sodium acetate.
3.9.2.81 Cystine (biochemical reagent). 3.9.2.9I),I-tryptophan (biochemical reagent). 3.9.2.10 10mol/L sodium hydroxide solution: weigh 200g sodium hydroxide and dissolve it in appropriate amount of water. Make up to 500ml. 3.9.2.11 (1+4) ethanol solution: mix 200ml anhydrous ethanol and 800ml water thoroughly. 3.9.2.12 Acid hydrolysis of casein: Weigh 50g vitamin-free casein into a 500mL beaker, add 200ml 3mol/l hydrochloric acid, and hydrolyze at 121(℃ for 6h. Transfer the hydrolyzate to evaporator III and evaporate it to a paste on a boiling water bath. Add 200ml water to dissolve it and evaporate it to a paste. Repeat this process 3 times to remove the hydrochloric acid. Use bromophenol blue as an external indicator and adjust the pH to 3.5 with 10mol/l sodium hydroxide solution. Add 20g activated carbon, shake, and filter. If the filtrate is not light yellow or colorless, it can be used Repeat the treatment with activated carbon. Dilute the filtrate to 500ml with water, add a little toluene on the liquid surface and store in the refrigerator. (This reagent can also be purchased from Dfico, product number No. 0288-15-6.) 3.9.2.13 Adenine, guanine, uracil solution: weigh 0.1g of adenine sulfate (purity 98%), guanine hydrochloride (biochemical reagent) and uracil in a 250mL beaker, add 75mL water and 2ml concentrated hydrochloric acid, then heat to completely dissolve. Cool. If there is precipitation, add a few drops of hydrochloric acid, heat again, and react. After cooling, dilute with water until no precipitation is produced, and store in refrigerator.
3.9.2.14 Vitamin solution 1: Weigh 25mg riboflavin, 25mg thiamine hydrochloride, 0.25mg biotin, 50mg niacin, dissolve in 0.02mol/l acetic acid solution and make up to 1000mL. 3.9.2.15 Vitamin solution: Dissolve 50mg p-aminobenzoic acid, 25mg calcium pantothenate, 100mg pyridoxine hydrochloride, 100mg pyridoxal hydrochloride, 20mg hydrochloric acid Pyridoxamine, 5mg folic acid are dissolved in (1+4) ethanol solution and the volume is adjusted to 1000mL. 3.9.2.16 A salt solution: Weigh 25g potassium dihydrogen phosphate and 25g potassium hydrogen phosphate and dissolve them in 500ml water, add 5 drops of concentrated hydrochloric acid and mix well. 3.9.2.17 E salt solution: Weigh 10g magnesium sulfate (MgSO47H,)), 0.5g sodium chloride, 0.5g manganese sulfate (MnS)·4H,)), 0.5g ferrous sulfate (FeSO.·7H0) and dissolve them in water and the volume is adjusted to 500mL, add 5 drops of concentrated hydrochloric acid and mix well. 3.9.2.18 Xanthine solution: Weigh 1.0g xanthine and dissolve it in 200mL water. Under heating conditions of 70C, add 30mL ammonium hydroxide (NH,OH) (2+3), stir until all solids are dissolved, cool and dilute to 1000mL with water. 3.9.2.19 Asparagine solution: weigh 1.0g L-asparagine and dissolve it in water, and dilute to 100ml. 3.9.2.20 Tween-80 solution: dissolve 25g Tween-80 in ethanol and dilute to 250mlL. 3.9.2.21 Vitamin Bz standard solution (all use brown reagent bottles). 3.9.2.21.1 Vitamin Bi2 standard stock solution (100ng/ml): weigh 50μg (balance with accuracy 0.01mg) of vitamin 3.2 dark red needle crystals, dilute to 500mL with (1+4) ethanol solution, and store in a refrigerator at 4C. 3.9.2.21.2 Vitamin B2 standard intermediate solution (1 ng/mL): Take 2.5 ml of the stock solution and dilute it to 250 ml with (1+4) ethanol. 4 (I transfer
Store in refrigerator.
GB/T 14924. 11 -- 2001
3.9.2.21.3 Vitamin B2 standard application solution (0.02 ng/mL): Take 1 ml of the intermediate solution and dilute it to 50 ml with water. Prepare it when needed. 3.9.2.22 Basic culture medium: The product of IDifco was used in the preparation of this standard, and the product number is No.0457-151. You can also prepare it yourself as follows:
Mix the following reagents in a 500mL beaker, add water to 200mL, use bromocresol purple as an external indicator, adjust the pH to 6.0~6.1 with 10mol/1. sodium hydroxide solution, and dilute to 250ml with water. Acid hydrolyzed casein
Adenine, guanine, uracil solution
Aspartyl solution
Yellow-80 solution
A salt solution
Ethyl salt solution
Vitamin solution
Vitamin solution I
Xanthine solution
Ascorbic acid
I-cystine
I). L-tryptophan
E. Glucose
Anhydrous sodium acetate
3.9.2.23 Agar medium: In 600mL water, add 15g protein, 5g water-soluble yeast extract powder, 10g anhydrous glucose. 2g anhydrous potassium dihydrogen phosphate. 100mL tomato juice, 10 mL Tween-80 solution, add 5.0-7.5g agar per 500ml liquid culture medium, heat to dissolve, adjust pH to 6.5-6.8 with 10mol/L sodium hydroxide, then make up to 1000mL, dispense into test tubes, sterilize at 121℃ (autoclave for 10min, take out and stand the test tubes upright, cool to room temperature and store in refrigerator. 3.9.2.24 Physiological saline: weigh 9.0g sodium chloride and dissolve in 1000ml water. Pour into 2-4 test tubes each time, add about 10mL to each tube, plug with cotton, sterilize at 121℃ (autoclave for 10min), and set aside. 3.9.2.25 0.4g/l bromocresol purple indicator: weigh 0.1g bromocresol purple in a small mortar, add 1. 6ml.0.1mol/l. sodium hydroxide, grind, add a little water and continue grinding until completely dissolved, dilute with water to 250mL. 3.9.3 Instruments and equipment
3.9.3.1 Common laboratory equipment.
3.9.3.2 Electric constant temperature incubator.
3.9.3.3 Pressure steam sterilizer.
3.9.3.4 Liquid rapid mixer.
3.9.3.5 Centrifuge.
3.9.3.6 Hard glass test tube: 20mm×150mm. 3.9.3.7 Spectrophotometer.
3.9.4 Preparation and preservation of strains and culture medium 3.9.4.1 Preparation of reserve strains: La ctobacillusleichmannii (ATCC7830) was inoculated in a straight agar culture tube, cultured in a (37±0.5)°C incubator for 16-24 hours, taken out and stored in a refrigerator, and subcultured at least once every two weeks. Subculture must be performed the day before the experiment.
3.9.4.2 Preparation of seed culture medium: Add 2mL 0.02ng/mL vitamin B,z standard working solution and 3mL basic culture medium to a 10ml centrifuge tube, plug it with a cotton plug, sterilize it at 121°C for 10 minutes, take it out and cool it, and store it in a refrigerator. Prepare two tubes each time and keep them for later use. 3.9.5 Operation steps
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GB/T 14924.11--- 2001
3.9.5.1 Preparation of inoculation solution: One day before use, inoculate L. leichmannii that has grown in agar tube for 16-24 minutes into seed culture solution, culture at (37±0.5)°C for 16-24 hours, take out and centrifuge at 3000r/min for 10 minutes, discard the supernatant, rinse twice with sterile saline, add 3mL of sterile saline, mix well, and pour the solution into a sterile syringe for inoculation. 3.9.5.2 Sample preparation
3.9.5.2.1 Preparation of hydrolyzate: Weigh 1.3g of anhydrous sodium dihydrogen phosphate, 1.2g of citric acid and 0.1g of sodium metabisulfite (NasS0), dissolve in water and make up to 100mL, prepare it when needed. 3.9.5.2.2 Weigh an appropriate amount of sample, place it in a 100mL conical flask, add 70ml of hydrolyzate, mix well, and sterilize at 121C for 10min. Take it out, cool it to room temperature, and filter it. Use bromocresol purple as an external indicator, adjust the pH to 6.0-6.1 with 10mol/L sodium hydroxide solution, transfer the hydrolyzate to a 100ml volumetric flask, and dilute it to the mark with water. The sample needs to be appropriately diluted so that the final concentration of NazS20s in the assay tube should be 0.03mg/ml.
3.9.5.3 Preparation of sample test tubes
Add 1.0, 2.0, 3.0, and 4.0ml of sample hydrolyzate to each set of parallel sample tubes, dilute it to 5mL with water, and then add 5ml of basic liquid culture medium.
3.9.5.4 Preparation of standard series tubes
Add 0.0, 1.0, 2.0, 3.0, 4.0, 5.0 ml of vitamin B2 standard working solution to each test tube. Make the content of vitamin B1 in each test tube to be 0.00 ng, 0.02 ng, 0.04 ng, 0.06 ng, 0.08 ng, 0.1 ng, add water to 5 ml. Then add 5 ml of basic liquid culture medium. Three sets of standard curves need to be made.
3.9.5.5 Sterilization: Both the sample tube and the standard tube are plugged with cotton plugs and sterilized at 121C for 10 minutes. Inoculation and culture: After the test tubes are cooled to room temperature, inoculate each tube with a drop of seed bacterial solution and culture in a (37±0.5)C constant temperature box for 16~20 hours. 3.9.5.6 Determination of absorbance: Under the condition of 640nm wavelength, adjust the instrument zero point with the zero tube in the standard series, and determine the absorbance value of the sample tube liquid and the standard tube culture.
3.9.6 Calculation
Use the ng number of the vitamin B12 standard series as the horizontal axis and the absorbance value as the vertical axis to draw a standard curve. Find the corresponding vitamin B12 content in the sample test tube on the curve based on the absorbance value in the sample test tube, and then calculate the vitamin B12 content in the sample according to formula (3):
6×100
m×1000
Where: X is the vitamin B12 content in the sample, μg/100g; - vitamin B12 content in the test tube, ng/mL; the fixed volume of the sample hydrolyzate, mL.;
factory. dilution multiple of the sample solution;
sample mass g.
3.9.7 Repeatability of results
The absolute value of the relative deviation of the results of repeated determination or simultaneous determination in the same laboratory is ≤10%. 3.10 Determination of vitamin K (menacetal) in compound feed 3.10.1 Principle
In the presence of ammonia, vitamin K (menacetal, Vk) forms a blue-purple colored substance with ethyl cyanoacetate. The absorbance value at 575nm is proportional to the concentration of vitamin K; The absorbance value of the colored substance is determined by a spectrophotometer, and the content of vitamin K in the sample is calculated from the standard curve. The detection limit of this method is 0.05mg. 3.10.2 Reagents
The reagents used in this experiment are all analytical grade, and the experimental water is distilled water. 3.10.2.10.1mol/L iodine solution: Weigh 25g potassium iodide and dissolve it in 20ml water, add 9.8g iodine reagent, mix well to dissolve, and add water to 75ml. Store in a brown bottle and keep away from light for 24 hours. GB/T 14924.11
3.10.2.20.1mol/L sodium thiosulfate (Na2S): Boil the water and then cool it. Weigh 25g sodium thiosulfate (Na2S) and dissolve it in 500ml of cooling water containing 0.1g sodium carbonate, and dilute it to 1000ml with cooling water. 3.10.2.3 Starch indicator: Weigh 2g soluble starch and add it to 10mL water, shake it. Then slowly add it to 200ml boiling water and boil for 2 minutes.
3.10.2.4 Ammonia-isopropyl alcohol solution: Mix isopropyl alcohol with an equal volume of concentrated ammonia. 3.10.2.5 Ethyl cyanoacetate (30g/L): Dissolve 3g ethyl cyanoacetate in 100ml isopropyl alcohol. 3.10.2.6 Vk standard solution (0.1 mg/mL) Accurately weigh 50 mg Vk standard and transfer it to a 500 ml brown volumetric flask. Dissolve it with isopropanol and make up to the mark.
3.10.3 Instruments and equipment
3.10.3.1 Common laboratory equipment.
3.10.3.2 Spectrophotometer.
3.10.4 Determination steps (all operations must be performed in the dark) 3.10.4.1 Extraction
Accurately weigh about 15 g of the mixed sample, accurately add 100 ml of water, stir for 10 minutes to ensure that Vk is fully dissolved and mixed. Filtration. If the filtrate is turbid, filter it repeatedly until it is clear. 3.10.4.2 Oxidation to remove impurities: Pipette 40 ml of filtrate into a 100 ml volumetric flask, add 1 to 2 drops of starch indicator, and use 0.1mol/1. iodine solution is titrated until a continuous blue color appears. Add 1 drop of 0.1mol/L Na2S.0 to the solution to eliminate the blue color. Make up to the mark with water. 3.10.4.3 Preparation of standard tubes and sample tubes: Take 2 sets of 20mL colorimetric tubes respectively, add V standard solution, sample extract and reagents in the order of Table 1, and prepare standard tubes and sample tubes. Table 1 Preparation of standard tubes and sample tubes
Vk standard solution, mL
Sample extract, mL
Isopropanol, mL
Ethyl cyanoacetic acid, mL
Ammonia-isopropanol, mL
Standard tube
Blank tube
Sample tube
Assay tube
Make up to 20mL with water, shake and place for 20min. 3.10.4.4 Colorimetric determination: Use a spectrophotometer at a wavelength of 575 cm, adjust the instrument zero point with a standard 0 tube, and determine the absorbance value of each tube. 3.10.5 Calculation: Use the standard Vz content as the horizontal axis and the absorbance value as the vertical axis, and draw a standard curve to calculate the regression equation. Use the difference in absorbance between the sample tube and the blank tube to find the Vk content of the sample tube on the standard curve, and then calculate the Vk content in the sample, see formula (4).
X = × 25 × 100
Where: X---m-the content of Vk in the sample, mg/100g; Ca-the V content corresponding to the difference in absorbance between the sample tube and the blank tube on the standard curve, mg; m--sample mass·g,
25-sample dilution multiple.
3.10.6 Permissible difference of results
The absolute value of the relative deviation of the results of parallel or repeated determinations in the same laboratory is less than 10%. 3.11 Determination of pantothenic acid in compound feed
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3.11.1 Principle
GB/T14924.11--2001
Pantothenic acid is an essential nutrient for the normal growth of Lactobucillus plantarum (ATCC8014). Under certain growth conditions, the growth and reproduction rate of Lactobucillus plantarum has a certain linear relationship with the content of pantothenic acid in the solution. The intensity of bacterial proliferation can be determined by turbidimetry or optical density method. By comparing with the standard curve, the pantothenic acid content in the sample can be calculated. The minimum detection limit of this method is 5ng. 3.11.2 Reagents
The water used in this test is distilled water, and all reagents used must be analytically pure reagents. 3.11.2.1 Toluene,
3.11.2.21mol/L hydrochloric acid solution.
3.11.2.3 Tris buffer solution: Dissolve 24.2g trishydroxyaminomethane in 150ml water, adjust the pH to 8.0-8.3 with 7.5mol/l sodium hydroxide solution, and then make up to 200ml, store in a 4C refrigerator, the shelf life is 2 weeks. 3.11.2.47.5mol/L sodium hydroxide solution: Dissolve 150g sodium hydroxide in water and make up to 500mL. 3.11.2.50.2mol/l acetic acid: Make up to 1000mL with 12ml glacial acetic acid. 3.11.2.6 0.2 mol/L sodium acetate solution: Dissolve 16.4 g sodium acetate in water and dilute to 1000 ml. 3.11.2.7 0.1 mol/L sodium bicarbonate solution: Weigh 0.85 g sodium bicarbonate and dissolve in water, then dilute to 100 ml. 3.11.2.8 0.2 mol/L potassium bicarbonate solution: Weigh 10.012 g potassium bicarbonate and dissolve in water, then dilute to 500 ml. 3.11.2.9 20 g/l alkaline phosphatase solution: Weigh 2 g alkaline phosphatase (Sigma No. P-3877) and dissolve in water, then dilute to 100 ml. Store in a 4°C refrigerator. 3.11.2.10 10% pigeon liver extract solution: Place the container used in a 4°C box overnight the day before preparing this reagent. (1) Weigh 30 g of pigeon liver acetone extract powder (Sigma No. I.-8376) and place it in a cold mortar. Add 300 ml of 0.2 mol/l potassium bicarbonate twice and grind it in an ice bath at 0°C to form a suspension. (2) Place the suspension in 8 centrifuge tubes, plug them tightly, shake them thoroughly, freeze them for 10 min, and then centrifuge them at 3000 r/min for 5 min. (3) Place the supernatant in a 500 mL cold beaker, add 150 g of ion exchange resin Dowex]-X8 (Bio-Rad Laboratories Inc., Brussels, Belgium), and shake them in an ice bath for 5 min. min; pour the mixture into a centrifuge tube and centrifuge at 3000/min for 5 min; (4) transfer the supernatant to another cold 500ml beaker and freeze for 10 min; (5) repeat steps (3) and (4) once; (6) then divide into test tubes and store under frozen conditions, thaw before use. 3.11.2.11 Acid hydrolysis of casein: weigh 50g casein without vitamins into a 500mL beaker, add 200ml 3mol/L hydrochloric acid, and hydrolyze at 121C under high pressure for 6h. Transfer the hydrolyzate to evaporator III and evaporate it on a boiling water bath until it is a paste. Add 200mL of water to dissolve it and evaporate it again until it is a granular state. Repeat this process 3 times to remove the hydrochloric acid. Use bromophenol blue as an external indicator and adjust the pH to 3.5 with 10mol/l sodium hydroxide. Add 2g of activated carbon, shake, and filter. If the filtrate is not light yellow or colorless, it can be treated with activated carbon again. Dilute the filtrate to 500ml with water, add a little toluene and store in a refrigerator. 3.11.2.12 Cystine and tryptophan solution: weigh 4g l-cystine and 1g L-tryptophan (or 2g DI-tryptophan) in 800mL water, heat to 70-80°C, add (1+5) hydrochloric acid dropwise, stirring continuously until completely dissolved. Cool to room temperature, dilute to 1000ml with water, store in a reagent bottle, add a little toluene on the liquid surface and store in a refrigerator. 3.11.2.13 Adenine, guanine, and uracil solutions: Weigh 0.1 g of adenine sulfate (purity 98%), guanine hydrochloride (biochemical reagent), and uracil in a 250 mL beaker, add 75 mL of water and 2 mL of concentrated hydrochloric acid, and heat to completely dissolve. Cool. If precipitation occurs, add a few drops of hydrochloric acid and heat again. Repeat this process until no precipitation occurs after cooling. Dilute to 100 mL with water. Add a little benzene to the liquid surface and store in a refrigerator.
3.11.2.14 Tween-80 solution: Dissolve 25 g of Tween-80 in ethanol and dilute to 250 mL. 3.11.2.15 Vitamin solution 1: Weigh 20 mg of riboflavin, 10 mg of thiamine hydrochloride, and 0.04 mg of biotin, dissolve in 0.2 mol/l acetic acid solution, and dilute to 1000 mL.
3.11.2.16 Vitamin solution II: 10 mg p-aminobenzoic acid, 50 mg niacin, 40 mg pyridoxine hydrochloride, dissolved in (1+3) ethanol lacquer solution, and fixed to 1000 ml. wwW.bzxz.Net
3.11.2.17 Salt solution A: Weigh 25 g potassium dihydrogen phosphate and 25 g potassium hydrogen phosphate, dissolve in 500 ml. water, add 5 drops of concentrated hydrochloric acid. 3.11.2.18 Salt solution B: Weigh 10 g magnesium sulfate (MgSO:·7H0), 1 g potassium chloride, 0.5 g manganese sulfate (MnS),·4H0) 2.1
GB/T 14924.11---- 2001
0.5g ferrous sulfate (FeS)·7H0), 23ml.85% phosphoric acid, dissolved in water and adjusted to 500ml3.11.2.19 Pantothenic acid standard solution
3.11.2.19.1 Pantothenic acid standard stock solution (40μg/ml): Weigh 43.47mg D-calcium pantothenate (Sigma Company. No.P.2250). Dissolve in 500mL water, add 10mL 0.2mol/L acetic acid, 100ml 0.2mol/l. sodium acetate, and then adjust the volume to 1000ml with water. At this time, the calcium pantothenate concentration of the solution is 43.47μg/ml, which is equivalent to a pantothenic acid concentration of 10μg/ml, and store in the refrigerator. 3.11.2.19.22 Pantothenic acid standard intermediate solution (1.0 μg/mL): Take 25 ml of the stock solution and add it to 500 ml of water, then add 10 ml of 0.2 mol/L acetic acid and 100 ml of 0.2 mol/L sodium acetate, then dilute to 1 000 ml with water and store in a refrigerator. 3.11.2.19.3 Pantothenic acid standard application solution (10 ng/ml): Take 1 ml of the intermediate solution and dilute to 100 ml with water and store in a refrigerator. 3.11.2.20 Basic culture medium: This standard uses a product from Difco, product number No. 0816-15-7. You can also prepare it yourself according to the following formula:
Mix the following reagents in a 500ml beaker and add water to 200mL. Use bromovanillin blue as an external indicator, adjust the pH to 6.8 with 10mol/L sodium hydroxide solution, and dilute with water to 250mL. Acid hydrolyzed casein
Cysteine, tryptophan solution
Adenine, guanine, uracil solution
Vitamin solution 1
Vitamin solution 1
Salt solution A
Salt solution B
Anhydrous glucose
Sodium acetate (NaAc·3H,O)
Tween-80 solution
3.11.2.21 Agar culture medium: Add 15g protein, 5g water-soluble yeast extract powder, 10g anhydrous glucose, 2g anhydrous potassium dihydrogen phosphate, 100ml tomato juice, 10ml. Tween-80, add 10-15g agar per 500ml liquid culture medium, heat and dissolve. Adjust pH to 6.5-6.8 with 7.5mol/l sodium hydroxide, then make up to 1000ml and dispense into test tubes. Sterilize at 121°C for 10 min, take out and place the test tube upright, cool to room temperature and store in refrigerator. 3.11.2.22 Physiological saline: Weigh 9.0g sodium chloride and dissolve in 1000ml water. Pour into 2~4 10ml test tubes each time, add about 10ml to each tube, plug with cotton, sterilize at 121°C for 10min, and set aside. 3.11.2.230.4g/1. Bromothymol blue solution: Weigh 0.1g bromothymol blue in a small mortar, add 1.6ml. 0.1mol/l. sodium hydroxide and grind, add a little water and continue grinding until completely dissolved, dilute with water to 250mL. 3.11.2.240.4g/l. Bromocresol green solution: weigh 0.1g of bromocresol green in a small mortar, add 1.4ml.0.1mol/L sodium hydroxide and grind, add a little water and continue grinding until it is completely dissolved, dilute with water to 250mL. 3.11.2.251g/l. Bromocresol blue ethanol solution: weigh 0.1g of bromocresol blue, dissolve it in ethanol, and dilute with ethanol to 100ml. 3.11.3 Instruments and equipment
3.11.3.1 Common laboratory equipment.
3.11.3.2 Electric constant temperature incubator.
3.11.3.3 Pressure steam sterilizer.
3.11.3.4 Liquid rapid mixer.
3.11.3.5 Centrifuge.
3.11.3.6 Spectrophotometer or turbidity meter. 3.11.3.7 Hard glass test tube: 20mm×150mm. 3.11.4 Preparation and preservation of bacterial strains and culture medium 3.11.4.1 Preparation of reserve bacterial strains: Lactobucillus plantarum (ATCC8014) was inoculated in a straight agar culture tube, and then incubated in a 37325
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