Phytic acid content detection kit micro method

Instruction manual for phytic acid content detection kit

Micro method

Item No.BC5845

Specifications100T/48S

Product composition: Before use, please carefully check whether the volume of the reagent matches the volume inside the bottle. If you have any questions, please contact the staff of Solaibao in a timely manner.

Preparation of solution:

1. Reagent 2: Add 4mL of Reagent 1 before use and dissolve thoroughly; Unused reagents are packaged and stored at -20 ℃4Zhou, avoid repeated freeze-thaw cycles;

2. Reagent 3: Dissolve 2.36mL of distilled water thoroughly before use, then slowly add the gun tip under the liquid surface0.64mLConcentrated sulfuric acid, thoroughly mixed; Can be stored at 2-8 ℃4week

3. Reagent 4: Dissolve 15mL of distilled water thoroughly before use, then slowly add the gun tip under the liquid surface15μLConcentrated sulfuric acid, thoroughly mixed; Can be stored at 2-8 ℃4week

4. Working fluid: Before use, according to the sample size, follow the ratio of reagent 3: reagent 4=1mL: 5mL (total)6mL，40T)Mix the ingredients evenly and use them up on the same day after configuration;

5. Standard: Dissolve 1.08mL of reagent thoroughly before use to prepare 10 μ mol/mL phytic acid standard, and store at 2-8 ℃ for 4 weeks;

6. Preparation of 250nmol/mL standard: Before use, mix 50 μ L of 10 μ mol/mL phytic acid standard with 450 μ L of distilled water to form 1 μ mol/mL standard (i.e. 1000nmol/mL); Take another 250 μ L of 1000nmol/mL standard substance and7Mix 50 μ L of distilled water to prepare a 250nmol/mL phytic acid standard, which is used for the determination of the standard tube in the following operation table. It is prepared and used immediately.

Product Description:

Phytic acid, also known as inositol hexaphosphate or cyclohexanol hexaphosphate, is widely present in eukaryotic cells and plays a key role in many cellular activities, such as maintaining inorganic phosphorus homeostasis, participating in plant hormone signal transduction, and serving as an enzyme cofactorDNARepairRNAEditor andmRNAOutput. Phytic acid provides the main source of phosphorus during plant germination and growth, and is widely present in plant-based foods such as grains, beans, fruits, vegetables, and dried fruits.

Under certain environmental conditions, phytase can decompose sodium phytate (disodium inositol hexaphosphate) to produce inorganic phosphorus and inositol derivatives. Under acidic conditions, inorganic phosphorus reacts with ammonium molybdate chromogenic reagent to produce a blue molybdenum blue substance, which has a characteristic absorption peak at 700nm. By measuring the content of inorganic phosphorus, the phytic acid content can be calculated.

Attention: It is recommended to select 2-3 samples with significant expected differences for pre experiments before the experiment.

Equipment and supplies to be provided:

Visible spectrophotometer/ELISA reader, low-temperature centrifuge, water bath/Constant temperature incubator, analytical balance, micro glass colorimetric dish/96Orifice plate, adjustable pipette, mortar/Homogenizerconcentrated sulfuric acid（>98%，AR）Ice and distilled water.

Operation steps:

1sample processing(The sample size to be tested can be adjusted appropriately, and the specific proportion can be referred to in the literature)

1. Fresh plant samples: by mass (g): one volume of extraction solution(mL)for1：5~10The proportion of (recommended to be around)0.1gJoin in1mLExtract solution 1) Add extract solution 1, homogenize thoroughly, and shake at room temperature2hAnd then4℃，10000gcentrifugation10min， 取0.8mLClear liquid, slowly add again0.15mLExtract solution 2, slowly blow and mix until no bubbles are produced,4℃ 10000gcentrifugation10minThen take the supernatant for testing.

2. Dry powder plant samples: by mass (g): one volume of extraction solution(mL)for1：5~20The proportion of (recommended to be around)0.05gJoin in1mLExtract solution 1) Add extract solution 1, homogenize thoroughly, and shake at room temperature2hAnd then4℃，10000gcentrifugation10min， 取0.8mLClear liquid, slowly add again0.15mLExtract solution 2, slowly blow and mix until no bubbles are produced,4℃ 10000gcentrifugation10minThen take the supernatant for testing.

3. Liquid sample: Take 100 μ L of liquid and add it1mLExtract solution one, shake at room temperature2hAnd then4℃，10000gcentrifugation10min， 取0.8mLClear liquid, slowly add again0.15mLExtract solution 2, slowly blow and mix until no bubbles are produced,4℃ 10000gcentrifugation10minThen take the supernatant for testing.

Note:Extract Liquid 2It needs to be added slowly as it will generate a large amount of bubbles. It is recommended to use it2Operate the mL EP tube.

2、 Measurement steps

1. Preheating of visible spectrophotometer/ELISA reader30minAbove, adjust the wavelength to700nmThe visible spectrophotometer is used to zero the distilled water.

2. Operation table: (Add the following reagents to a 1.5mL EP tube)

3、 Calculation of phytic acid content

1. Calculate based on sample protein concentration

Phytic acid content（nmol/mg prot）=ΔA determination×Cmark÷ΔA standard×Vsample÷(Vsample×Cpr) =250×ΔA determination÷ΔA standard÷Cpr

2. Calculated based on sample quality

Phytic acid content（nMol/g mass)=ΔA determination×Cmark÷ΔA standard×(VShangqing+VExtract Liquid 2)÷(W×VShangqing÷VExtraction solution one)

=296.875×ΔA determination÷ΔA standard÷W

3. Calculated by liquid volume

Phytic acid content（nmol/mL）=ΔA determination×Cmark÷ΔA standard×(VShangqing+VExtract Liquid 2)÷[Vliquid×VShangqing÷(VExtraction solution one+Vliquid)]

=3265.625×ΔA determination÷ΔA standard

C standard: standard tube concentration, 250nmol/mL； V sample: Add sample volume,0.12mLV supernatant: volume of supernatant during extraction,0.8mL；VExtraction solution 2: Add the volume of extraction solution 2,0.15mL；VExtraction solution one: volume of added extraction solution one,1mLCpr: sample protein concentration,mg/mLV liquid: volume of liquid sample,0.1mLW: Sample quality,g.

Notes:

1. If∆A measurement is less than0.010Alternatively, if the absorbance value of the measuring tube is close to that of the blank tube, the sample size can be increased before proceeding with the measurement; if∆A measurement is greater than1It is recommended to dilute the sample supernatant with distilled water before conducting the measurement. Pay attention to synchronously modifying the calculation formula.

2. If the sample becomes turbid after adding the working solution, it is recommended to dilute the sample supernatant with distilled water before conducting the measurement. Pay attention to synchronously modifying the calculation formula.

3. Extract 1 contains protein precipitant, therefore the supernatant cannot be used for protein concentration determination. To determine protein content, a separate sample needs to be taken.

Experimental Example:

1. Weigh 0.1044g of onion sample, follow the extraction and measurement steps, and use96Calculation of orifice plate measurement:ΔA determination=Adetermination-Acomparison=0.411-0.375=0.036，ΔA standard=Astandard-Ablank=0.811-0.096=0.715Calculated based on sample quality:

Phytic acid content（nMol/g mass)=296.875×ΔA determination÷ΔA standard÷W=143.18nMol/g mass.

2. Weigh 0.05g of wheat flour and follow the extraction and measurement steps96Calculation of orifice plate measurement:ΔA determination=Adetermination-Acomparison=0.331-0.188=0.143，ΔA standard=Astandard-Ablank=0.811-0.096=0.715Calculated based on sample quality:

Phytic acid content（nMol/g mass)=296.875×ΔA determination÷ΔA standard÷W=1187.5nMol/g mass.

References:

[1] Senna R, Simonin V, Silva-Neto M A C, et al. Induction of acid phosphatase activity during germination of maize (Zea mays) seeds. [J]. Plant Physiology & Biochemistry, 2006, 44(7-9):467-473.

[2] Iqbal T H, Lewis K O, Cooper B T. Phytase activity in the human and rat small intestine[J]. Gut, 1994, 35(9):1233-1236.

[3] Azeke M A, Egielewa S J, Ihimire E. Effect of germination on the phytase activity, phytate and total phosphorus contents of rice (Oryza sativa), maize (Zea mays), millet (Panicum miliaceum), sorghum (Sorghum bicolor) and wheat (Triticum aestivum)[J]. Journal of Food Science&Technology, 2011.

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Phytic acid content detection kit micro method Phytic acid content detection kit micro method