HPLC Method for Inulin Ceriabelgium

Underground Fat Loss Manual

Best Weight Loss Programs That Work

Get Instant Access

To supply the manufacturers with an appropriate means for production, food labeling, and quality control of inulin and inulin-containing products, the merits of an enzymatic digestion method followed by HPLC have been tested for the quantitative assessment of inulin. Moreover, if total soluble dietary fiber is wanted, the standard AOAC method has to be modified by including an enzymatic treatment with inulinase in addition to the amyloglucosidase incubation step in order to remove the residual P-fructans from the soluble fiber fraction (Figures 1 and 2). In these conditions these P-fructans directly determined have to be added to the ''soluble fiber fraction'' obtained with the AOAC modified method.

Enzymatic Digestion

Inulin can be hydrolyzed chemically or enzymatically to yield fructose and glucose (7). In the enzymatic digestion step, Novozym SP 230® Novo Nordisk enzyme hydrolyzes inulin totally. As shown in Figure 1, the enzymatic digestion yields the total amount of fructose, which is then quantified by HPLC (R1).

A blank test is run to account for any free fructose (R2) and the result is substracted from the value of the assay. To compute the inulin equivalent, (R1-R2) is multiplied by the correction factor 1.1 (assuming that the ratio of fructose/ glucose is 85/15 in inulin). Since inulin is a natural product, this ratio fluctuates somewhat in practice according to the origin of the raw product from which inulin is obtained.

For samples containing sugar (sucrose), a correction has to be made since the Novozym SP 230® enzyme also totally splits sucrose, producing 50% glucose and 50% fructose. This problem can be alleviated by analyzing a duplicate sample of identical weight and followed likewise by HPLC analysis, the fructose originating from sucrose (R3) can thus be assessed and substracted also from the amount of fructose (R1) obtained by the Novozym SP 230® digestion. This method gives the P-fructan dietary fiber (see Equations (1) and (2)).

Removal of P-Fructans

This is usually achieved with Novozym SP 230® inulinase after the treatment with amyloglucosidase. This procedure yields the total dietary fiber content except for P-fructan dietary fiber.

Dysseler et al.

Amyloglucosidase Diagram
Figure 1 P-Fructan determination

Materials and Methods: P-Fructan Dietary Fiber Method

Apparatus ^


High pressure pump, Millipore Waters (type 510) !fj>

Automatic sample injection device, Waters (type 712) <!

Oven with temperature control, Millipore Waters TCM 5

Refractometer detector, Millipore WATERS (type R401) with thermostatic J

water bath (30°C) for the refractometer cell a

Integrator, Millipore WATERS (type 740) |

HPLC column Hypersil APS NH2 (Chrompack), 20 cm g

HPLC working parameters |

Mobile phase: Water-acetonitrile: 15%-85% (V/V)

Inulin and Oligofructose Determination

Inulin and Oligofructose Determination

Hplc Inulin
Figure 2 Flow diagram of the AOAC dietary fiber method (991.43: Lee et al., Ref. 5) modified for total dietary fiber to assure total removal of p-fructans

Flow rate: 0.4 ml/min. Column temperature: 30°C

Test Samples

1. Standards: fructose, glucose, saccharose and sorbitol MERCK GLC grade (calibration samples)

2. Inulin

3. Inulin (for biochemical use, Merck n°4733)

4. Fibrulin (Cosucra company) batch 1

Dysseler et al.

Fibrulin (Cosucra company) batch 2 Raftiline® (Raffinerie Tirlemontoise) Raftilose® (Raffinerie Tirlemontoise) Yogurt

Commercial samples, dosed with known amounts of inulin Plain yogurt from skimmed milk and yogurt from skimmed milk with fruit ingredients


Duplicate samples are required for this analysis:

1. Sample to be digested by Novozym SP 230®

2. Blank sample

Hydrolysis of fructans by Novozym SP 230® enzyme

1. Weigh accurately a sample of ca 10 g containing ca 1 g ''Fructan'' into 400 ml beaker

2. Add 150 ml acetate buffer solution 0.1 M, pH = 4.5

3. After homogenizing, cover beaker with aluminium foil. Place the beaker in the shaking water bath at 85°C for 20 minutes, to dissolve the sample

4. Remove the beakers from the bath and cool down to 60°C

5. Add 100 |l Novozym SP 230® enzyme and incubate in shaking water bath at 60°C for 30 minutes for total digestion of the inulin

6. Cool to room temperature and transfer quantitatively to 200 ml volumetric flask

7. Add 3 ml of lead hydroxy acetate to precipitate the protein and make up to

8. 200 ml with water

9. After homogenizing, filter through filter paper

10. Transfer a small amount of the solution into a capped vial

11. Carry out the HPLC assay with this solution.

12. The quantity of fructose obtained by HPLC (R1) represents the total amount of fructose in the sample.

13. Weigh accurately (within 20 mg) a duplicate sample of ca 10 g of the sample used for enzyme digestion; proceed exactly in the same manner as for the test sample, but without addition of Novozym SP 230®.

The quantity of fructose obtained by HPLC (R2) represents the amount of free fructose in the sample. (R3) represents the amount of fructose obtained in the sample by HPLC.

Inulin and Oligofructose Determination

Calculation and Expression of Results

The amount of ''fructan dietary fiber'' (FDF) is given by:

% P-FDF = 11 (R1 R2) 100 (1) for samples containing no sucrose, and o/n™. 1.1 [R1 - (R2 + R3)] 100

% n-FDF =-^-----(2) for samples containing sucrose,


R1 = concentration of the total fructose (g/l) R2 = concentration of the free fructose (g/l) R3 = concentration of the fructose (g/l) from sucrose P = mean mass (g/l) of the duplicate test samples,where P = 5 times the mean dry mass of the test sample taken

Empirical conversion factor for fructose to fructan, assuming a ratio of 80 to 85% fructose and 15% glucose in the fructan polymer.

Modified A.OA.C. Dietary Fiber Method 991.43 Without P-Fructan Dietary Fiber

As shown in Figure 2, we apply A.O.A.C. TDF method 991.43 (Lee et al., Ref. 5), but with some modification. This minor modification includes only a stage of inulinase digestion performed, after the amyloglucosidase digestion. It aims to eliminate the fructans, under the following conditions:

inulinase: Novozym SP 230® 1800 inu/g, 100 ml temperature: 60°C; pH: 4.5

The pH adjustment step for the inulinase is the same as for amyloglucosidase. g


Inulin Assay—Samples Without Sugar g

Inulin samples were digested with Novozym SP 230® to check the performance |

of the hydrolysis according to the HPLC procedure for samples without sugar. As shown in Table 1, the enzymatic digestion recovery is higher than 95%.

The moisture contents of all the samples, as determined separately, were ca 5%. The correction factor applied to compute the amount of inulin in the sample varies between 1.0 and 1.2 for the different fructans. However, in practice, a correction factor 1.1 for routine analyses should procure fairly accurate results.

Dysseler et al.

Table 1 Analysis of Inulin and Oligofructose Samples With and Without Added Sugar












fructans (g/l)


Samples without sugar

Inulin (Merck)




5.19 (0.29)






4.83 (0.35)






4.82 (0.38)






4.98 (0.31)






4.71 (0.28)


Sugar-dosed samples

Fibrulin-1 + sugar





3.35 (0.14)


*Indicates industrial product

The analytical data are mean values of four series of analyses each, the confidence limits are computed for a probability 0.05.

*Indicates industrial product

The analytical data are mean values of four series of analyses each, the confidence limits are computed for a probability 0.05.

Inulin Samples Containing Sugar

The influence of the presence of sugar has been checked on one of the industrial inulin samples. To this effect, 60% Fibrulin-1 was dosed with 40% sucrose, weighed accurately.

The digestion procedure was carried out with Novozym SP 230® according to the given procedure. The recovery is close to 100% (as in Table 1). Thus, the interference of sugar can be considered as negligible for practical purposes.

Inulin Assay with Yogurt

After having ascertained the suitability of the Novozym SP 230® for the assessment of fructans, inulin was assessed on commercial yogurt samples dosed with known amounts of inulin.

Two commercial yogurt samples, (ca 150 g) plain yogurt and yogurt, which contains sugar sweetened fruit, were each blended homogeneously with 6.6% of accurately measured inulin (ca 10 g). This represents ca 40% inulin on dry matter basis. The yogurt with fruit contains ca 4% sugar. The data shown in Table 2

Table 2 Analysis of Inulin in Yogurt












fructans (g/l)


Plain yogurt





3.03 (0.09)


Fruit yogurt





2.88 (0.12)

Inulin and Oligofructose Determination

are mean values of 4 analyses. The results show that for plain yogurt (which contains only very small amounts of sucrose) the recovery is 101%, a very good result for practical purposes. For fruit yogurt (where the sucrose content is significantly higher) the 96% recovery value is also quite acceptable.

Was this article helpful?

0 0
Quick Weight Loss Action

Quick Weight Loss Action

Why Indulge In Self-Pity When You Can Do Something About Your Weight Now. Say Goodbye to Your Weight Problems That Have Only Make Your Life Nothing But Miserable. Have you often felt short-changed because of your weight or physical appearance?

Get My Free Ebook

Post a comment