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L. Prosky Associates, Rockville, MD 20850 and * Orafti Analytical Service, B-3300 Tienen, Belgium
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • methodology • inulin • oligofructose • fructan
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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and the USDA, have defined dietary fiber as the
material that precipitates in 78% ethanol by the AOAC International
official methods for dietary fiber analysis (AOAC 1990
, 1992a
,
1992b
and 1994
). In 1992, the Codex Alimentarius
Commission endorsed two methods for the analysis of dietary fiber, AOAC
985.29 for total dietary fiber in special foods and AOAC 991.43 for
total dietary fiber in infant formula and follow-up formula
(Codex 1992
). It was known that there were smaller
polysaccharides that did not precipitate completely at this
concentration of alcohol, and it remained for scientists to prove that
the unprecipitatible material behaved physiologically like dietary
fiber and further to devise a method that would measure this entity in
a successful AOAC collaborative study.
As a follow-up to a previously held International Life Science
Institute (ILSI North America 1994
) mini-workshop on
complex carbohydrates held in Washington, DC the previous year, a
workshop was held at the AOAC International meeting in Nashville in
1995 to determine if there was agreement among representative
scientists as to a definition of complex carbohydrates and dietary
fiber (AOAC International Workshop 1995
). There was
general agreement among the workshop participants that dietary fiber
should be included in the definition of complex carbohydrates, but more
importantly, they agreed that resistant oligosaccharides, namely,
inulin and oligofructose, be included in the dietary fiber complex. The
results of three AOAC International surveys also supported the
expansion of the dietary fiber definition to include resistant
oligosaccharides (Lee and Prosky 1994 and 1995
).
Further, representatives of the FDA in December of 1995 stated that
they would consider inulin and oligofructose as dietary fiber if the
method for their measurement would pass the scrutiny of an AOAC
International collaborative study.
Fructans are mixtures of molecules consisting of fructose moieties
linked to each other by ß(2
1) bonds. Glucose molecules may be
linked to the end of the chain by an 
(12) bond as occurs in
sucrose. The degree of polymerization (DP) varies from two to several
hundred, with the major components of fructans being inulin (mostly DP
2–60) and oligofructose (DP 2–10). These two components occur in
significant amounts in many fruits and vegetables.
The principle of the method was first published by Quemener et al. 1994
and has since been refined at Orafti Analytical
Service (De Leenheer and Hoebregs 1994
, Dysseler et al. 1993
, Van Loo et al. 1995
). The method
relies on the enzymatic treatment of products with an inulinase
(fructozym), followed by determination of the released sugars
(Hoebregs 1997
).
Fructans are first extracted from the product with boiling water. An aliquot of this extract is treated with amyloglucosidase. A part of the hydrolyzate is treated with inulinase; glucose, fructose and sucrose are assayed in the first and second hydrolyzates and in the initial sample by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The concentration of fructans is calculated by the difference in these determinations.
Because the oligofructose is not recovered in the AOAC-total dietary
fiber (TDF) methods, and a small fraction of inulin is
recovered by the AOAC-TDF methods (Coussement 1995
),
a correction should be made so that the inulin would not be counted
twice. Inulinase may be added to the enzyme complex in the TDF methods
removing all fructans from the sample (Quemener et al. 1996
). One can also determine the fructan content in
precipitates of the TDF methods and subtract this amount from the TDF
amount. This paper describes the method and results obtained in a
successful AOAC International collaborative study held during
1996–1997 (Hoebregs 1997
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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This method depends on the enzymatic treatment of a food product with
an inulinase enzyme (fructozym), followed by determination of the
released sugars. The fructans are extracted from the product with
boiling water. An aliquot of the extract is hydrolyzed with
amyloglucosidase and a part of the hydrolyzate is treated with
fructozym. The first and second hydrolyzates and the initial sample are
analyzed using HPAEC-PAD. In sugar analysis 1, free fructose
(Ff) and sucrose (S) are
determined in the initial sample. In sugar analysis 2, the sum of the
amount of free glucose (Gf) and glucose from
maltodextrins and starch (Gm) are determined
in the first hydrolyzate (with amyloglucosidase). In sugar analysis 3,
the total amount of glucose (Gt) and the
total amount of fructose (Ft) are determined
in the second hydrolyzate (with fructozym). The concentration of
glucose and fructose released from fructan is calculated by the
difference in these determinations. Glucose released from fructan is
expressed as follows:
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Fructose released from fructan is expressed as follows:
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The fructan content (i) is the sum of
Gi and Fi,
corrected for water loss during hydrolysis as follows:
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Apparatus and materials.
The materials used included the following: analytical balance, sensitivity ± 0.1 mg, pH meter, temperature compensated, standardized with pH 4.0, 7.0, and 9.0 buffer solutions; glass bottles, 100 or 150 mL, with screw caps obtained from Duran Schott (Mainz, Germany) or equivalent; water baths capable of maintaining 85 ± 2°C and 60 ± 2°C and with shaking capability; high performance anion-exchange chromatograph including one Dionex (Sunnyvale, CA) 4500i LC gradient pump or equivalent, equipped with an eluent degas module, a microinjection valve, a pulsed electrochemical detector working in a pulsed amperometric detection mode (PAD) or equivalent, an automated sampler and an HPAEC column Carbopac PA1 pellicular anion-exchange resin (250 x 4 mm) with a Carbopac PA guard column (50 x 4 mm) or equivalent; data integrator from Shimadzu (Kyoto, Japan), C-R4 AX Chromatopac or equivalent; membrane filters from Schleicher & Schuell (Dassel, Germany), 0.2 µm, Spartan 30/A or equivalent; glass microfiber filters, Whatman GF/F, 25 mm i.d., (Catalog No. 1825 025, Whatman International, Maidstone, UK) or equivalent; filter holder, 25 mm i.d., for low pressure syringe or equivalent; vacuum oven capable of maintaining 55 ± 3°C; dessicator with SiO2 or equivalent, biweekly, dry dessicant overnight at 130°C; column oven, Nooreg 100 column oven series 2, (No. 090199, Dionex Corporation, Sunnyvale, CA) or equivalent; vortex mixer, Labinco (Breda, The Netherlands) type L24, or equivalent.
Reagents.
For sample preparation and extraction and for mobile phase reagents preparation, use water quality Type 1, reagent grade (ASTM or ACS standards for purity) throughout. Acetate buffer, pH 4.5, 28 mL of 0.2 mol/L acetic acid and 22 mL of 0.2 mol/L Na acetate are mixed with water and made up to 100 mL. Lyophilized amyloglucosidase, A-7420, containing <0.02% glucose, fructose, and sucrose was obtained from Sigma Chemical (St. Louis, MO). This should be kept refrigerated when not in use. Inulinase solution (Fructozym) containing <0.005% glucose, fructose, and sucrose was obtained from Novo Industry (Copenhagen, Denmark). Store enzyme solution in refrigerator when not in use. Carbonate-free NaOH solution, density 1.54, 50% NaOH (J. T. Baker, No. 7067, Mallinckrodt Baker, Phillipsburg, NJ) or equivalent was used; this should be stored under He for stability reasons. The HPAEC mobile phase A was carbonate-free 10 mmol/L NaOH prepared by degassing 2 L water in a bottle of the Dionex degas module for at least 15 min with N2 or He. Without shaking or mixing the 50% NaOH solution and while blowing He on the liquid surface in the bottle, pipette 1.04 mL from the middle of the bottle and add gently to the degassed water. Continue degassing for 30 min before using. HPAEC mobile phase B was carbonate-free 1 mol/L NaOH. This was prepared in the same way as A but 109.5 mL 50% NaOH solution was added to 2 L degassed water instead of 1.04 mL. Continue degassing the solution for 30 min before using. Other reagents include the following: glucoheptose, D-glucoheptose (No. G-11, Pfanstiehl Laboratories, Waukegan, IL), or equivalent; lactose, D(+)-lactose monohydrate (Ph. Eur., puriss. p.a. Maltitol, Sigma, M-8892, ~98%, Sigma Chemical), or equivalent; glucose, fructose, sucrose and galactose, reagent grade. The preparation of standardized sugar stock solution (SS) is as follows. Dry reference sugar standards, except lactose and maltitol, in a vacuum oven at a temperature set to maintain 55 ± 3°C for 48 h. Weigh and transfer into a 100-mL volumetric flask 100 mg of each of the following reference sugar standards: maltitol, dextrose, fructose, lactose, galactose and sucrose. Allow to dissolve completely in water by using a magnetic stirrer bar. Make up to 100 g by weight. Preparation of internal standard stock solution (IS) is as follows. Weigh and transfer 100 mg glucoheptose standard into a 100-mL volumetric flask. Allow to dissolve completely in water by using a magnetic stirrer bar. Make up to 100 g by weight. Preparation of working standards is as follows: 1) Use 50 mg/kg glucoheptose, maltitol, dextrose, galactose, lactose, fructose and sucrose. Dilute 5.0 g IS and 5.0 g of SS to 100 g with water (S1). 2) Use 50 mg/kg glucoheptose, 25 mg/kg dextrose, maltitol, galactose, lactose, fructose and sucrose. Dilute 5.0 g IS and 2.5 g SS to 100 g with water (S2). 3) Use 50 mg/kg glucoheptose, 5 mg/kg dextrose, maltitol, galactose, lactose, fructose and sucrose. Dilute 5.0 g IS and 0.5 g SS up to 100 g with water (S3).
Analytical procedures.
Sample preparation. Homogenize samples immediately before analysis. For fruits, vegetables, cereal products and processed food products, mix with interruptions in a blender for 2–10 min. Freeze sticky or fatty products such as chocolate and bars that form a paste-like mass in the blender before mixing in the blender. Cut gummy and sticky products that cannot be mixed in a blender into small pieces with a knife or scissors so that no particle is larger than 100 mm3. Hard samples like candies should be shattered in a mortar so that no particle exceeds 100 mm3.
Extraction.
See Figure 1
for flow diagram of fructan method. Weigh an amount of sample
(preferably containing ~1 g fructan, but not exceeding 30 g
sample and/or 5 g starch) accurately to ± 0.1 mg in a 100-mL
beaker containing a stirring rod (M1). See
guide values in Table 1.
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Because of the possible existence of insoluble matter and/or fat in some sample extracts, it may be difficult to homogenize them well. Therefore, the following procedure is recommended for taking subsamples: shake volumetric flask containing sample extracts vigorously, transfer contents of flask to a beaker and continue vigorous mixing in the beaker (e.g., by using a magnetic stirrer); using a Pasteur pipette (e.g., a plastic one with a large tip), take the subsample while continuing the vigorous mixing.
Samples containing fructans in solution are diluted to ~1% fructan solution without any heating.
Enzymic hydrolysis. Keep one aliquot of ~50 g aside for direct analysis (assay A0). Transfer ~15 g of homogenized mixture (M3) accurately weighed to ±10 mg in a tarred glass bottle with screwcap; add the same amount of acetate buffer to adjust the pH to 4.5 ± 0.05. If necessary, adjust with 0.05 mol/L KOH or 0.05 mol/L HCl.
Add a sufficient amount of amyloglucosidase, taking into account the amount of starch and maltodextrins present in the sample and the concentration of the enzyme (e.g., for Sigma's amyloglucosidase with 51 U/mg, use 1 mg enzyme for 50 mg starch, because 1 U will liberate 1 mg of glucose from starch). If the amount of starch and maltodextrins is unknown, consider the unknown part of the sample as 100% starch. As guide values for paste-like samples, use 35 mg amyloglucosidase (with 51 U/mg); for other samples use 10 mg. For starchy products from which 5 g of sample has been extracted in 250 mL, also use 10 mg.
Incubate the mixture for 30 min at 60 ± 2°C with constant, mild agitation. The heating time should be 30 min from the time the reaction mixture reaches 60°C. The shaking should be such that no foam forms and no air bubbles are brought into suspension. Allow to cool to room temperature and weigh (net weight is M4). Keep one aliquot of ~10 g aside for analysis (assay A1).
To the remaining part of the first hydrolysate (net weight is M5), add a sufficient amount of fructozym, taking into account the amount of fructan present in the sample and the concentration of the enzyme (e.g., for Novo's fructozym with 1.8 U/mg, use 56 mg of enzyme for 100 mg fructan, because 1 U will hydrolyze 1 mg of fructan; guide value, 150 mg fructozym). If the amount of fructan is unknown, consider the unknown part of the sample as 100% fructan.
Incubate again for 30 min at 60 ± 2°C with constant, mild agitation. The heating time should be 30 min from the time the reaction mixture reaches 60°C. The shaking should be such that no foam forms and no air bubbles are brought into suspension. Allow to cool to room temperature and weigh (net weight is M6; assay A2).
HPAEC-PAD determination of mono- and disaccharides. The chromatographic conditions are as follows: analytical column, Carbopac PA1, 4.0 mm i.d. x 25 cm, or equivalent; guard column, Carbopac PA, 4.0 mm i.d. x 5 cm, or equivalent; column temperature, 40 ± 0.5°C; mobile phase A, carbonate-free 10 mmol/L NaOH; mobile phase B, carbonate-free 1 mol/L NaOH; flow rate, 1.0 mL/min; injection volume 50 µL; detector, pulsed electrochemical detector in PAD mode; detector sensitivity, analog range 1–3°C. Note: parameters may vary in order to optimize the chromatography.
Sample preparation for HPAEC-PADF analysis.
Dilute samples from assays A0,
A1 and A2 so that
the concentrations of glucose, fructose and sucrose are within the
concentration range of the working standards and add internal standard
glucoheptose. Dilute 2.0 g IS stock solution and an amount of
sample assay A0
(M7) to 100 g with water. Dilute
2.0 g IS stock solution and an amount of
A1 (M8) to
100 g with water. Dilute 2.0 g IS stock solution with an
amount of sample assay A2
(M9) to 100 g with water. The amounts
of sample assays A0,
A1 and A2 depend
on the type of product to be analyzed. Find guide in Table 1
.
For food samples with a fat content more than five times the fructan content, defat the diluted samples before injection.
Transfer ~4 g of diluted solution into a reaction tube and add 4 mL of hexane. Shake for 5 min on a vortex and centrifuge to separate the organic phase. Discard the organic phase with a Pasteur pipette. Repeat extraction with 4 mL of hexane, centrifuge and discard organic phase. Filter samples through Whatman GF/F glass microfiber filters and through 0.2 µm membrane filters before injection.
Note: prepare two different dilutions of the same sample solution if expecting a large difference between concentrations of different sugar compounds to be analyzed.
Sample analysis. Run the three calibration standards first to establish the linearity of the system. Repeat the three standards and between each standard run two samples (e.g., [S1, samples 1A0 and 1A1], [S2, samples 1A2 and 2A0], [S3, samples 2A1and 2A2], [S1, samples 3A0 and 3A1], [S2 and so on]. Continue until all samples have been chromatographed.
Use average response factors from the standards bracketing the samples to calculate sugar concentrations for each sample.
Integration. Peak width, threshold settings and other integration parameters should be chosen to ensure that the same type of integration is used for both samples and standards. Carefully control the baseline selection. Use peak height or peak area for quantitation.
Possible interferences. For samples containing maltitol and/or lactose, such as some chocolates, choco paste, cheese and breakfast drinks, the glucose results after hydrolysis are possibly overestimated. Therefore, also calculate the maltitol amounts in samples and correct glucose results for that part of maltitol that has been hydrolyzed by fructozym. Also correct glucose results for the galactose that has been formed by the hydrolysis of lactose by fructozym.
Calculations.
Calculate response factors for fructose, glucose, sucrose, maltitol and
galactose with the following formula:
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where R is the response factor; C is the concentration of sugar standard in mg/kg; C' is the concentration of IS glucoheptose in mg/kg: h is the height of sugar standard peak; and h' is the height of the IS peak.
With reference to Table 2, calculate the fructose and sucrose contents in the diluted
samples from assay A0, the glucose concentration
in the diluted samples from the assay A1 and the
fructose and glucose content in the diluted samples from assay
A2.
|
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where C is the concentration of sugar in mg/kg;
R is the response factor; C' is the concentration
of IS in mg/kg in the diluted sample; h is the height of
sugar peak; and h' is the height of the IS peak. Calculate
free fructose (Ff) and sucrose contents
(S), glucose (G1), maltitol
(Mal1), and galactose
(Gal1) contents after hydrolysis with
amyloglucosidase, and total glucose (Gt),
and total fructose (Ft) and maltitol
(Mal2) and galactose
(Gal2) contents after hydrolysis with fructozym
in percentage of initial sample with the following equations:
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where CFf is the mg fructose/kg
diluted solution in assay A0.
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where Cs is the mg sucrose/kg
diluted solution in assay A0.
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where CG1 is the mg glucose/kg
diluted solution in assay A1.
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where CMal 1 is the mg maltitol/kg
diluted solution in assay A1.
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where CGal1 is the mg galactose/kg
diluted solution in assay A1.
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where CGt is the mg glucose/kg
diluted solution in assay A2.
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where CFt is the mg fructose/kg
diluted solution in assay A2.
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where CMal2 is the mg maltitol/kg
diluted solution in assay A2.
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where CGal2 is the mg galactose/kg diluted solution in assay A2.
Fructan calculation. Calculate
Gi and Fi
with the following equations:
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where Gi is the glucose from fructans; GS is the glucose released from sucrose (S/1.9); GMal is the glucose released from maltitol [(Mal1 - Mal2)/1.9]; GLac is the glucose released from lactose (Gal2 - Gal1); Fi is the fructose released from fructans; and FS is the fructose released from sucrose (S/1.9).
The fructan content (i) is calculated according to the
following equation:
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where k = [180 + 162(n - 1]/180n; and n (the average DP) = [(Fi/Gi) + 1] for pure GFn mixtures. For inulin from chicory, n = 10 can be used (k = 0.91); for oligofructose, n = 4 can be safely used (k = 0.925).
Note: the amount of glucose formed by the hydrolysis of lactose may be calculated by dividing the difference in lactose content before and after the fructozym hydrolysis by 1.9. The amount of glucose formed by the hydrolysis of maltitol is the same as the amount of sorbitol formed by the hydrolysis of maltitol with fructozym.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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3). Only three laboratories followed the standardized procedures
exactly. All others either decreased the frequency of analysis of
standard solutions or performed the standardization in a separate
series. The result was that the fluctuation of response intensity of
the detector with time caused greater inaccuracies in the results.
One pair of data from laboratory 3 was eliminated by the Cochran test
because of unacceptable variation between replicates. Pair 6 from
laboratory 3 was eliminated by the Grubb's test because of the
extremely high average compared with the values reported by the other
collaborators. Repeatability relative standard deviation
(RSDr) values ranged from 2.8 to 5.6% and
reproducibility relative standard deviation
(RSDR) values ranged from 5.6 to 13.0%. All
results from one laboratory that performed analysis on an aminopropyl
silica column and refractive index (RI) detector (instead of the
prescribed anion-exchange column and PAD) were higher except for
the chocolate sample, which was lower. When statistical analysis was
performed without the results from this laboratory, analytical results
(dry ice mix and biscuits) from another laboratory were eliminated by
the Grubb's test, the first because of extremely low average, the
second because of extremely high average. Final results of the
statistical analysis are shown, with and without outliers, in
Table 4. RSDr values ranged from 2.9 to 5.8% and
RSDR values ranged from 4.7 to 11.1%.
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On the basis of the results obtained in this collaborative study, the ion-exchange chromatographic method for fructan determination in food and food products was adopted as a first action by the AOAC International. Results of AOAC International surveys and workshops strongly suggest that resistant oligosaccharides such as inulin and oligofructose be included in the dietary fiber complex. Further, inulin and oligofructose have been accepted as fiber in 12 countries (Austria, Belgium, Denmark, France, Germany, Greece, Ireland, Italy, Netherlands, Norway, Portugal and Switzerland) with approvals pending in six countries (U.K., U.S.A., Spain, Sweden, Canada and Australia). On the basis of these findings, the natural occurrence of these fructans in vegetables and fruits, and the physiologic effects associated with inulin and oligofructose, I, as General Referee for Dietary Fiber and Complex Carbohydrates of the AOAC International, conclude that inulin and oligofructose are indeed part of the dietary fiber complex.
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FOOTNOTES |
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3 Abbreviations used: DF, dietary fiber; DP,
degree of polymerization; FDA, Food and Drug Administration;
HPAEC-PAD, high performance anion-exchange chromatography with
pulsed amperometric detection; IS, internal standard stock solution;
PAD, pulsed amperometric detection; RI, refractive index; RSDr,
repeatability standard deviations; RSDR, reproducibiliity
standard deviations; SS, standardized sugar stock solution; TDF, total
dietary fiber.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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1. Association of Official Analytical Chemists (1990) AOAC Method 985.29. Total dietary fiber in foods: enzymatic-gravimetric method. Official Methods of Analysis,15th ed. JAOAC International, Arlington, VA.
2. Association of Official Analytical Chemists (1992) AOAC Method 991.42. Insoluble dietary fiber in foods and food products: enzymatic-gravimetric method, phosphate buffer. Official Methods of Analysis,15th ed., 3rd suppl. JAOAC International, Arlington, VA.
3. Association of Official Analytical Chemists (1992) AOAC Method 991.43. Total, soluble, and insoluble dietary fiber in foods: enzymatic-gravimetric method, MES-TRIS buffer. Official Methods of Analysis, 15th ed., 3rd suppl. JAOAC International, Arlington, VA.
4. Association of Official Analytical Chemists (1994) AOAC Method 993.19. Soluble dietary fiber in foods and food products: enzymatic-gravimetric method, phosphate buffer. Official Methods of Analysis, 15th ed., 5th suppl. JAOAC International, Arlington, VA.
5. Association of Official Analytical Chemists International Workshop (1995) Definition and analysis of complex carbohydrates/dietary fiber. Proceedings of the AOAC Worshop on Complex Carbohydrates and Dietary Fiber. Nashville, TN, September 1995.
6. Codex Committee on Methods of Analysis and Sampling (1992) 18th Session, ALINORM 93/23.
7. Coussement, P. (1995) Inulin and oligofructose as dietary fiber: analytical, nutritional and legal aspects. Proceedings of the AOAC Worshop on Complex Carbohydrates and Dietary Fiber. Nashville, TN, September 1995.
8. De Leenheer L., Hoebregs H. Progress in the elucidation of the chicory inulin. Stache/Starke 1994;46:193-196
9. Dysseler, P., Hoffem, D., Fockedey, J., Quemener, B., Thibault, J. F. & Coussement, P. (1993) Determination of inulin and oligofructose in food products (AOAC dietary fiber modified method). Proceedings of the COST 92 Workshop, October 21 and 22, Marseille, France.
10. Hoebregs H. Fructans in foods and food products, ion-exchange chromatographic method: collaborative study. J. Assoc. Off. Anal. Chem. Int. 1997;80:1029-1037
11. International Life Science Institute North America. (1994) Complex carbohydrates. Washington, DC.
12. Lee S. C., Prosky L. Perspectives on new dietary fiber definition. Cereal Foods World 1994;39:767-768
13. Lee S. C., Prosky L. International survey on dietary fiber: definition, analysis, and reference materials. J. Assoc. Off. Anal. Chem. Int. 1995;78:22-36
14. Quemener B., Thibault J. F., Coussement P. Determination of inulin and oligofructose in food products, and integration in the AOAC method for measurement of total dietary fiber. Lebensm.-Wiss. Technol. 1994;27:125-132
15. Quemener, B., Thibault, J. F. & Coussement, P. (1996) Integration of inulin determination in the AOAC method for measurement of total dietary fiber. Plant Polysaccharides Symposium, July 17–19, Abstract No. 11, Nantes, France.
16. Van Loo J., Coussement P., De Leenheer L., Hoebregs H., Smits G. On the presence of inulin and oligofructose as natural ingredients in the Western diet. Crit. Rev. Food Sci. Nutr. 1995;35:525-552[Medline]
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