果聚糖检测试剂盒 Fructan Assay Kit 货号：K-FRUC Megazyme试剂盒

发表于由
果聚糖检测试剂盒

英文名：Fructan Assay Kit

货号：K-FRUC

规格：100 assays per kit

市场价： 4800元

产品名称：果聚糖检测试剂盒
英文名称：Fructan Assay Kit
型号规格：100次
品牌：爱尔兰Megaeyme公司

果聚糖广泛分布于植物界。单子叶植物，双子叶植物和绿藻中均存在。

以分子结构和分子重量区别果聚糖。也可以分为三类：菊粉组，果聚糖组和带支链组。菊粉组主要或专门由果糖基果糖键组成。果聚糖组主要或专门由果糖基果糖键组成。带支链组由大量的（2→1）和（2→6）果糖基果糖键共同组成。

目前存在很多测定植物材料和食品中果聚糖的方法。被广泛接受的是水解为D-果糖（和D-葡萄糖）后再测定的方法。这存在将蔗糖，D-果糖和D-葡萄糖分别去除或测定的问题，Pontis(1966)已经报道了蔗糖，D-葡萄糖和D-果糖的去除方法，利用蔗糖酶水解蔗糖，再通过氢氧化钠煮沸破坏随之产生的D-果糖和D-葡萄糖及原有的单糖。据报道，蔗糖酶对菊粉组低聚果糖的作用是缓慢的，可以通过选择正确的孵育时间使其失效。通过测定目前使用的纯化的酵母蔗糖酶，发现很难。

但是，本试剂盒提供的方法操作简单，使用标准实验室设备，并且是精确性，可重复性和专一性都很好。采用的酶是高纯度酶，专一性水解蔗糖，淀粉和果聚糖。采用的蔗糖酶快速水解蔗糖但对1-蔗果三糖和其他果聚糖的水解活性可以忽略不计（McCleary和Blakeney,1999）(图2)。在底物浓度为10 mg/mL时,蔗糖与1-蔗果三糖的相对水解比率为3,800：1。

原理：

蔗糖被蔗糖酶水解为D-葡萄糖和D-果糖。同时，如果样品中存在淀粉和麦芽多糖通过高纯β-淀粉酶，普鲁兰酶和麦芽糖酶联合作用被水解D-葡萄糖。用碱性***物处理生成的还原糖，将其继续被还原为糖醇。用稀释的乙酸将溶液调至中性，并去除过量的***物。果聚糖被纯化果聚糖酶（菊粉外切酶）水解为D-葡萄糖和D-果糖，通过PAHBAH法测定生成的还原糖。这个方法方便操作，D-葡萄糖和D-果糖的对应颜色一致。含有半乳糖苷蔗糖寡糖的样品如（棉子糖）在蔗糖/淀粉酶混合物（酶溶液1）处理前推荐使用α-半乳糖苷酶（来源于黑曲霉）孵育。释放的单糖通过碱性***物处理去除。如果处理中包括这一步骤，需要在计算中考虑体积的变化（如溶液5的最终体积应用1.15mL代替1.1mL）.

 

分析物意义：许多食品如洋葱和种子中的常见组分

Megazyme检测试剂盒优点：方法新颖、反应快、试剂稳定 

The Fructan test kit is suitable for the specific measurement and analysis of fructan in plant extracts and food products containing starch, sucrose and other sugars.

Colourimetric method for the determination of Fructan in plant
products, foodstuffs and other materials

Principle:
(sucrase)
(1) Sucrose + H2O → D-glucose + D-fructose

(β-amylase + maltase + pullulanase)
(2) Starch + maltosaccharides + H2O → D-glucose

(borohydride)
(3) D-Glucose + D-fructose → D-sorbitol + D-mannitol
(non-reducing)

(exo-inulinase + endo-inulinase)
(4) Fructan + H2O → D-glucose + D-fructose

(100°C, 6 min)
(5) D-Glucose + D-fructose + PAHBAH → PAHBAH colour complex

Kit size:                            100 assays
Method:                            Spectrophotometric at 410 nm
Total assay time:              ~ 90 min
Detection limit:                 1-100% of sample weight
Application examples: 
Flours, plant materials (e.g. onion), food products and other materials
Method recognition:     
AOAC (Method 999.03), AACC (Method 32-32.01) and CODEX 
(Type III Method)

Advantages

  • Very cost effective
  • All kit reagents stable for > 2 years after preparation
  • Unaffected by high sucrose / reducing sugar concentrations
  • Fructan kits are only available from Megazyme
  • Simple format
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing
  • Standard included

Q1. Should the pH of the sample be adjusted even for samples in acidic media?

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.
Samples above 0.1 mL are more likely to affect the pH of the assay solution and therefore the pH of these samples should be adjusted as described in the data booklet, prior to addition to the assay.

Q2. Does the fructanase enzyme hydrolyse Neosugar?

Yes.

Q3. When measuring fructan in starch containing samples, i.e. maize, is there a possibility of incomplete starch hydrolysis in step B?

From our experience, the level of beta-amylase/pullulanase etc. added is more than adequate to completely hydrolyse any starch likely to be present.  However, if there is a suspicion of incomplete starch hydrolysis, you can check this by running the hydrolysis with the pullulanase/beta-amylase etc. over a range of incubation times and comparing the results.

Q4. Could you tell me whether this method simply gives a gross measure of the fructan content, or whether it is possible to specifically determine the inulin content of the material?

The method measures inulin and cereal fructans.

Q5. What typical absorbance values are obtained for fructan control and fructose control?

Fructan Control Flour (29.6%) 0.496 0.498
Fructose Control 1.133 1.135.

Q6. What is the precision of the method for measurement of fructans?

Our reproducibility is usually +/- 5 to 7% of the fructan value.

Q7. Do you have statistics for the variability of your diagnostic test kits? I would like to get an idea what typical %RSD values would be for your oligofructan test kit.

With all our kits we can obtain approx. 5% c.v. in interlaboratory studies.  In-house these values are closer to 3%.

Q8. Can I detect the levan content of some plant materials by use of your fructan kit?

The fructanase in the kit will completely hydrolyse the fructans in plants.  These are either inulin type or "branched" type (as in oat or wheat stems).  We do not know if the enzymes would act on pure levan.

Q9. What actually is the factor value (F)? Is it always 54.5 or does it change depending on the fructose content?

The value of 54.5 is the actual amount of fructose standard used.  This amount of fructose will give a certain colour in the PAHBAH assay.
Thus; F = factor = [54.5 (micrograms of fructose)] divided by the absorbance value obtained for 54.5 micrograms of fructose in the PAHBAH method.

Q10. Is it possible to buy Fructanase separate from the kit?

Yes.  The best enzyme to use for analytical purposes is exo-Inulinase (E-EXOIAN).

Q11. We need to analyse sucrose in extracts of chicory roots and the sucrase used in your fructan assay procedure may solve the problem. Do you think that it would be possible to use sucrase instead of invertase in the sucrose assay?

Yes, the sugar enzyme can be used to measure sucrose.  The enzyme is an alpha-glucosidase, so it will also hydrolyse maltose if this is present.

Q12. Is it possible to do the weighing and hot extraction the day before the actual assay?

Extract solutions are best made and analysed the same day (not stored overnight). But if they are stored overnight in a refrigerator, heat them to 80˚C, and cool before analysing (to ensure all fructan has re-dissolved).

Q13. Can the fructan method be used to measure inulin in starch-containing products, i.e. Cornflakes?

Yes.

Q14. There is an issue with the performance of the kit; the results are not as expected.

If you suspect that the Megazyme test kit is not performing as expected such that expected results are not obtained please do the following:

  1. Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
  2. Send the results of the kit standard, blank samples and the results obtained for your sample,  in the relevant MegaCalc spreadsheet (if available) to Megazyme (cs@megazyme.com). Where available the relevant MegaCalc spreadsheet can be downloaded from where the product appears on the Megazyme website.
  3. State the kit lot number being used (this is found on the outside of the kit box).
  4. State which assay format was used (refer to the relevant page in the kit booklet if necessary).
  5. State exact details of any modifications to the standard procedure that is provided by Megazyme.
  6. State the sample type and describe the sample preparation steps if applicable.

Q15. Sucrase is specific for the cleavage of sucrose without hydrolysing any of the other potentially present oligo/polysaccharides (mixed linked glucan, soluble starch, fructan of any size & structure). Is this correct? Do you sell the enzyme separately?

Yes , the sucrase does hydrolyse sucrose only and has no action on the other carbohydrates that you mention.  The “sucrase” enzyme is an alpha-glucosidase which we sell as Alpha-Glucosidase (Maltase) E-MALTS.

Q16. Fructan Assay Procedure “Further treatment of extracts” Step 3, Pg 8 "if sample is cooled, it must be re-heated to 80˚C to ensure fructans redissolve". If frozen to –20˚C, thawed quickly & re-heated to 80˚C, will the fructans re-dissolve completely?

Fructans are very soluble and will redissolve very readily.  They may precipitate on freezing (at least the polymeric portion) but they will easily re-dissolve at 80˚C.

Megazyme 溶解淀粉 操作视频

Megazyme 试剂盒样品前处理准备操作视频

Megazyme果聚糖检测试剂盒Fructan操作视频（K-FRUC）