果聚糖檢測(cè)試劑盒
英文名：Fructan Assay Kit
貨號(hào)：K-FRUC
規(guī)格：100 assays per kit
產(chǎn)品名稱：果聚糖檢測(cè)試劑盒
英文名稱：Fructan Assay Kit
型號(hào)規(guī)格：100次
品牌：愛爾蘭Megaeyme公司
果聚糖廣泛分布于植物界。單子葉植物，雙子葉植物和綠藻中均存在。
以分子結(jié)構(gòu)和分子重量區(qū)別果聚糖。也可以分為三類：菊粉組，果聚糖組和帶支鏈組。菊粉組主要或?qū)ｉT由果糖基果糖鍵組成。果聚糖組主要或?qū)ｉT由果糖基果糖鍵組成。帶支鏈組由大量的（2→1）和（2→6）果糖基果糖鍵共同組成。
目前存在很多測(cè)定植物材料和食品中果聚糖的方法。被廣泛接受的是水解為D-果糖（和D-葡萄糖）后再測(cè)定的方法。這存在將蔗糖，D-果糖和D-葡萄糖分別去除或測(cè)定的問題，Pontis(1966)已經(jīng)報(bào)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，再通過(guò)氫氧化鈉煮沸破壞隨之產(chǎn)生的D-果糖和D-葡萄糖及原有的單糖。據(jù)報(bào)道，蔗糖酶對(duì)菊粉組低聚果糖的作用是緩慢的，可以通過(guò)選擇正確的孵育時(shí)間使其失效。通過(guò)測(cè)定目前使用的純化的酵母蔗糖酶，發(fā)現(xiàn)很難。
但是，本試劑盒提供的方法操作簡(jiǎn)單，使用標(biāo)準(zhǔn)實(shí)驗(yàn)室設(shè)備，并且是精確性，可重復(fù)性和專一性都很好。采用的酶是高純度酶，專一性水解蔗糖，淀粉和果聚糖。采用的蔗糖酶快速水解蔗糖但對(duì)1-蔗果三糖和其他果聚糖的水解活性可以忽略不計(jì)（McCleary和Blakeney,1999）(圖2)。在底物濃度為10 mg/mL時(shí),蔗糖與1-蔗果三糖的相對(duì)水解比率為3,800：1。
原理：
蔗糖被蔗糖酶水解為D-葡萄糖和D-果糖。同時(shí)，如果樣品中存在淀粉和麥芽多糖通過(guò)高純β-淀粉酶，普魯蘭酶和麥芽糖酶聯(lián)合作用被水解D-葡萄糖。用堿性***物處理生成的還原糖，將其繼續(xù)被還原為糖醇。用稀釋的乙酸將溶液調(diào)至中性，并去除過(guò)量的***物。果聚糖被純化果聚糖酶（菊粉外切酶）水解為D-葡萄糖和D-果糖，通過(guò)PAHBAH法測(cè)定生成的還原糖。這個(gè)方法方便操作，D-葡萄糖和D-果糖的對(duì)應(yīng)顏色一致。含有半乳糖苷蔗糖寡糖的樣品如（棉子糖）在蔗糖/淀粉酶混合物（酶溶液1）處理前推薦使用α-半乳糖苷酶（來(lái)源于黑曲霉）孵育。釋放的單糖通過(guò)堿性***物處理去除。如果處理中包括這一步驟，需要在計(jì)算中考慮體積的變化（如溶液5的最終體積應(yīng)用1.15mL代替1.1mL）.
分析物意義：許多食品如洋蔥和種子中的常見組分
Megazyme檢測(cè)試劑盒優(yōu)點(diǎn)：方法新穎、反應(yīng)快、試劑穩(wěn)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:
- Ensure that you have tested the standard sample that is supplied with the Megazyme test kit.
- 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.
- State the kit lot number being used (this is found on the outside of the kit box).
- State which assay format was used (refer to the relevant page in the kit booklet if necessary).
- State exact details of any modifications to the standard procedure that is provided by Megazyme.
- 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.