VitaminB3

Vitamin B3 Test Kit(Pantothenic acid)REAGEN™Sample and Vitamin B3 culture medium add to the 96-microwell which has the Lactobacillus plantarum subsp.

  • Brand:REAGEN
  • Model:RNF91030
  • Specifications:96T
  • Price:837.00USD

Vitamin B3 Test Kit(Pantothenic acid)


REAGENSample and Vitamin B3 culture medium add to the 96-microwell which has the 

Lactobacillus plantarum subsp. lactisleichmannii,if the sample hasVitamin B3 the Lactobacillus 

plantarum will grow,after culture 37,44-48h, read the OD by microplate reader 610 - 630 nm 

(or540 - 550 nm),The sample of the turbidity compared with standard curve can get Niactin content.


Product Description


REAGENSample and Vitamin B3 culture medium add to the 96-microwell which has the Lactobacillus plantarum subsp. lactisleichmannii,if the sample hasVitamin B3 the Lactobacillus plantarum will grow,after culture 37,44-48h, read the OD by microplate reader 610 - 630 nm (or540 - 550 nm),TREAGENVitamin B3 Test Kit is quantitative analysis of total Vitamin B3 in food ,feed and drug.

 The unique features of the kit are:

rapid, and cost-effective extraction methods,the same as other Vitamin.

The kit provide positive control and serial standards

High sensitivity (20μg/100g)

High recovery90%-105%

High reproducibility

he sample of the turbidity compared with standard curve can get Niactin content.


Kit Contents, Storage and Shelf Life

REAGEN Vitamin B3 Test Kit has the capacity for 96 determinations or testing of 42 samples 

in duplicate (assuming 12 wells for standards). Return any unused microwells to the foil bag and 

reseal them with the desiccant provided in the original package. Store the kit at 2-8 .The shelf 

life is 6 months when the kit is properly stored.



Kit Contents

                                   Amount

Storage

Lactobacillus plantarum -Coated Plate

1 x 96-well Plate (8 wells x 12 strips)

2-8

Sterile water

3x30m

2-8

Vitamin B3 Culture medium

32-8

Vitamin B3 standards

32-8

Aluminum foil

22-8

Required Materials Not Provided With the Kit

 

lLaminar flow bench

lMicrotiter plate reader610 - 630 nm 540 - 550 nm

lIncubator37 °C

lWater bath 95

lCentrifuge8000g

lSterile Pipette 20-200ul,100-1000ul

lSterile centrifuge tubes (1.5-2ml  ,15 and 50 mL)

lThe 500 mL glass bottles and flasks (100 and 1000 mL), 100 mL beaker

lSterile syringe, 0.2 um sterile membrane

lSterile water or deionized water

lAmylasezB. Fluka 86250

lH2SO41mol/L

lNaOH concentrate(take 40g NaOH add 100ml Sterile water or deionized water)

    and 2mol/L NaOH(take 8g NaOH add 100ml Sterile water or deionized water)

lNaOH1mol/L and 0.1mol/L

lHCl1mol/L and 0.1mol/L

lCitrate bufferpH4.5,take 1.5 g citric acid monohydrate to 100 ml beaker , add 50 ml sterile water or deionized water, stirring and dissolved, then add 12 ml NaOH (1 mol/L) or 0.48 g NaOH, using 0.1 mol/L HCl to pH to 4.5;With sterile water or deionized water transferring quantitative solution into 100 ml volumetric flask, with sterile water or deionized water constant volume, can be stored three days in 2-8 .


Sample Preparation


Add the Niactin detection solid fortified food, need hot water extracted under alkaline conditions;Add 

Niactin , test liquid samples should be in sterile filtration and sterile water diluted for testing.The total 

Niactin content in samples, the samples must be using enzymes for processing.Samples should be 

stored at 4  avoid light.Standard and samples should be in triplicate.Samples of unknown samples

 should be extract twice dilution.Take liquid should day using samples, and should be stored away from light.


The Sample Extract Preparation


Add 1g sample to 40 ml sterile water or deionized water or extract solution ,the diluted multiples is 40.Diluted multiples directly included in the standard curve.For the lower concentration of vitamin samples, sample quantity should be increased to 5 g (mL) (results should be taken into consideration).The following sample must be sterile filtered or sterile extraction

1) The sample extraction without heating, such as juice or health drinks (in 95  water bath heating except for 30 min sample).

2) Samples containing traditional Chinese medicine (TCM) and seasoning and honey and tea

3) Vitamin mixture, premix or tablets (sample higher levels of vitamin B3)  (in 95  water bath heating except for 30 min sample).

4) The vitamin content low dark sample (filtration process can remove color).

5) If the sample contains solid particles or sample turbidity effect filter, in front of the sterile filtration process should be in more than 8000 g centrifugal 5 min.

Note: If the sample in the 95  heating 30 min, do not need to be sterile filtration.But the sample extract kit provides no bacteria of water must be used for dilution.


The Sample Extract Diluted


1) Dilution ratio calculation

To a concentration of 2500μg/100g as an example of a solid sample, with the concentration of divided by the concentration of the standard 2 get diluted multiples.

Dilution multiples = 2500μg/40μg= 62.5.

So we know the sample need to dilute about 1:60 with sterile water.

2) Dilution steps

A)110 (100μl sample extraction solution + 900μl sterile water)
B) 16(100μl A + 500μl sterile water)
Note: Each step dilution need fully blending and then take the next step diluted, extract the diluent should now match with now.

The Sample Processing


1) Liquid Samples

Add 1mL sample to 50mL sterile centrifuge tupe,add 40mL 1x sample extraction buffer,vortex,sterile filtration.(or 95 water bath 30mins,quickly cool to below 30).If the concentration is high,dilute in the 1.5-2ml sterile tube.

2) Pectinose and Sweets

Take 15 to 20 g pectinose or sweets to 50 mL sterile centrifuge tube, add about 40 mL sterile water or deionized water, dissolve the sample in 95  water bath.Quickly cooling below 30 , with sterile water or deionized water transferring quantitative extraction solution into 100 mL flask, with sterile water or deionized water constant volume.Take about 1 g sample extraction solution to 50 mL sterile centrifuge tube, fill with sterile water or deionized water to 40 mL, oscillation, sterile filtration (or the sample in 95  water bath heating 30 min, and then quickly cooled to below 30 ). If the concentration of Vitamin B3 is high,dilute in the 1.5-2ml sterile tupe.Such as pectinose samples of 17 g , then transferred to the solution of sterile centrifuge tube  (1 g sample corresponding to extract solution) for volume: 100 mL/17g = 5.88 mL/g

3) Capsules, pills and vitamin mixture

The first ,determine the weight of each capsule or pill (weighing 5 capsules or pills, average), and then put the pills in a mortar or blender crush (capsules can be extracted directly when cut) .Capsules, pills and vitamin mixture content of Niactin usually is higher, if the dilution ratio (100 g of biotin content in the samples divided by the standard 2 content of  Niactin content of Niactin more than 10000, suggest an extraction in advance before the sample preparation (with 1000 ml sterile water or deionized water)

4)   Diluted multiples of 20000 sample preparation and extraction

Take 1 g pills, vitamins mixture or cut the capsule to 500 ml glass bottles, add about 400 ml sterile water or deionized water, shake .With HCl or NaOH dispatch pH to 8.0±0.2. In 95  water bath extraction 30 min, there should be at least 5 times, and then quickly cooling below 30 .With sterile water or deionized water transferring extraction solution to 1000 ml flask and constant volume with sterile water or deionized water.Transferring extraction solution 1 mL to 50 mL sterile centrifuge tube, fill with sterile water or deionized water to 40 mL, shake, sterile filtration (or the sample in 95  water bath heating 30 min, and then quickly cooled to below 30 ).1:20 dilute in the 1.5-2ml sterile tube with sterile water.

Note: the results of calculation should be considered when diluted multiples is2000, dilution steps will fill 1 ml to 40 ml has been included in the standard curve.

5)  Grain, baby food, bread, flour and dairy products

Take 1 g sample to 50 ml sterile centrifuge tube, add 40 ml sterile water or deionized water,shake. in 95  water bath extraction 30 min, should be at least 5 times (make sure that the centrifugal tube seal), and then quickly cooling below 30 .Using 0.2 um sterile membrane filtration to 1.5 or 2.0 mL sterile reaction tube.According to the Vitamin B3 concentration range, then use of sterile water to filtrate further dilution.

1)  Total Vitamin B3

Take 1g(mL) sample to 50 ml sterile centrifuge tube, add 20 ml sterile water or deionized water,shake. Dispatch pH to 4.5 with HCl (or using citrate buffer instead of sterile water extract samples, add 1 g sample to 20 ml pH4.5 citrate buffer, shake, need not dispatch pH).Add 300 mg of amylase, shake, 37  avoid light 1 h incubation (incubator or water bath pot), intermittent shake at the same time.Fill with sterile water or deionized water to 40 ml, in 95  water bath heating 30 min, there should be at least 5 times (make sure that the centrifugal tube seal), and then quickly cooling below 30 .Centrifuga l5 min under greater than 8000 g, according to the concentration range of Niactin, at 1.5 or 2.0 mL sterile water used in the reaction tube of supernatant further dilution.

7) The total Niactin in yeast and yeast products

Take about 1 g sample to 50 ml sterile centrifuge tube, add 30 ml 1 m H2SO4 , shake .In the 95  water bath extraction 30 min, should be at least 5 times (make sure that the centrifugal tube seal), and then quickly cooling below 30 , with the concentrate of NaOH and 2 mol/L NaOH dispatch pH to 6.0-7.0.Fill with sterile water or deionized water to 40 mL, shake, the sterile filter ( in 95  water bath extraction30 min, and then quickly cooling below 30 , centrifugal), according to the concentration range of  Niactin, at 1.5 or 2.0 mL sterile water used in the reaction tube of supernatant further dilution.


Test protocol


All the reagents need to return to room temperature.shake the reagents before use. Don't pollute the kit’s reagents.

Add 2 mL sterile water to standard , cover cap and shake points dissolved with standard is the standard concentrate.

Filter the standard to 2mL tube by 0.2um sterile filter.

Prepare standard: Sterile filtration the concentrate standard.

standard curve μg/100g(ml)

Add Water   uL


Add standard  uL


Total volume uL

            

 0

1000

+

0

=

1000

 1:20

990

+


10

=

1000

 2:40

980

+

20


=

1000

 3:80

960

+

40 


=

1000

 4:160

920

+

80 


=

1000

5:400

800

+

200


=

1000

 Vitamin B3 Culture medium preparation

Use tweezers to take out the desiccant medium cap and open and discarded.

Add 10 ml sterile water to Culture medium,lid and mix

Water bath 95 5mins and shake some times,quickly cool to room temperature.

Filter the culture medium to 15mL tube by 0.2um sterile filter.


Testing process

 

1. All the protocols’ water must be sterile

2. Get out some wells ,put back the left wells to the foil bag and seal.

3. Get 150uLculture medium to the wells

4. Add 150uL standards or samples to the wells

5. Get out Aluminum foil paper ,remove the protect layer of the foil paper,seal the wells By Pressing the foil paper on the wells(must be sealed).

6. Incubation 37,44-48h,in dark.

7. Shake the plate and let the Microorganism dissolve in the culture medium.(or press the foil paper and reverse the plate on the desk ,shake to dissolve the Microorganism)

8. Slowly take off  the foil paper from the plate wells.not let the solution splash.

9. Break all the foams in the wells by Pipette tips.

10. Read OD value by micro plate reader 610-630nm(or 540-550nm) .

Notice:It is better read the OD after incubation. This plate can be store at 2-8 just for 48h but not open the foil paper.

Validity


Blank well OD<STANDARD 1  OD

STANDARD 5  OD>0.6

Standard curve :vertical coordinate: OD;Horizontal coordinate :concentration.

Read the sample concentration on the curve. If extra dilute the sample ,Multiply the dilution factor.

Warnings and Precautions


1) Kit should be 2-8  storage, do not use when expired.
2) To join in microwell plate sample extract or diluent must be sterile, other consumables for the  experiment must also be sterile.
3) Test medium can stimulate eye, skin and mucous membrane, should be used with caution.
4) After the completion of the test should be carried out in accordance with the regulations on waste treatment (such as high pressure sterilization).

Kinds of Vitamin test kit for food sample

Cat #

Product

Sensitivity

Sample Type

Amount

RNF91030

Vitamin B3(Niacin)

200μg/Kg

Grain, infant food, flour, milk

96 T

RNF91029

Vitamin B5(Pantothenic acid)

800μg/Kg

Grain, infant food, flour, milk

96 T

RNF91023

Vitamin B7 (Biotin)

0.4μg/Kg

Grain, infant food, flour, milk

96 T

RNF91021

Vitamin B9(Folic acid)

0.5μg/Kg

Grain, infant food, flour, milk

96 T

RNF91022

Vitamin B12(Cyanocobalamin)

0.6μg/Kg

Grain, infant food, flour, milk

96 T



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REAGEN LLC,focusing on ELISA kits including clinical disease detecting kits,food&feed safety test kits and animal epidemic virus detecting kits in US for 6 years

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