Handbook of Vanilla Science and Technology

Determine absorbance of vanillin solution at 270,348, and 380 nm using the blank solution to zero the machine at each wavelength.

Calculate absorbance of 1 ppm vanillin as follows:

Absorbance (corrected) = (A₃₄₈ - 0.29A₂₇₀ + 0.71A₃₈₀)/3

A₃₄₈ is the peak of absorption of vanillin. Background absorption from polyphenolic and other interfering compounds in the sample is corrected for by subtracting a percentage of the absorbance at 270 and 380 nm.

Standard Vanillin Factor (SVF) = Absorbance (corrected) × 10. The SVF should be in the range of 1.5 to 1.54.

9.2.4.3 Vanillin determination in vanilla extracts and other vanilla products

See Table 9.3 for using appropriate amounts for analysis for different folds of extracts and for using appropriate multiplier for calculating vanillin. The following procedure is given for one-fold vanilla extract.

Pipet 5 ml vanilla extract in a 100 ml volumetric flask and bring to volume with distilled water. (Solution A)

Pipet 2 ml Solution A into another 100 ml volumetric flask and add 2 ml 0.1 N NaOH solution and bring to volume with distilled water. (Solution B)

Pipet 2 ml Solution A into another 100 ml volumetric flask and bring to volume with distilled water. (Solution C)

Table 9.3 Sample preparation for vanillin determination

Vanilla Product | To make solution A Use | To make solution B Use | 0.1N NaOH Use | Multiplier Use

1-Fold Extract | 5 ml | 2 ml | 2 ml | 1

2-Fold Extract | 5 ml | 2 ml | 2 ml | 1

3-Fold Extract | 5 ml | 1 ml | 2 ml | 2

4-Fold Extract | 5 ml | 1 ml | 2 ml | 2

5-Fold Extract | 2 ml | 1 ml | 2 ml | 5

10-Fold Extract | 1 ml | 1 ml | 2 ml | 10

Oleoresin | 0.1 g (wt) | 2 ml | 2 ml | 50

Determine the absorbance of Solution B at 348 nm using a spectrophotometer and using Solution C as a reference solution to zero the machine.

Vanillin content in the extract is calculated as:

A₃₄₈ (Multiplier from the table)/Standard Vanillin Factor

9.2.4.4 HPLC method

This method gives a more accurate estimate of vanillin content of a vanilla product. Vanilla extracts (and other vanilla products) show lower values for vanillin when analyzed using the HPLC method than when the UV spectrophotometric method is used. This is because in HPLC analysis, interference from other vanilla components, such as p-hydroxybenzalde-hyde and p-hydroxybenzoic acid, is eliminated. Typical system parameters are as follows:

I The liquid chromatograph instrument consists of a solvent delivery system, sample injector valve or auto sampler and a light absorbance detector with UV detection set at 254 nm;

II Column: stainless steel with dimensions of 100 × 4.6 mm to 250 × 4.6 mm packed with 5 to 10 pm particle size C8 stationary phase;

III Solvents: LC grade methanol, LC grade water, 95% ethanol and glacial acetic acid;

IV Mobile phase is methanol-acidified water (10 + 90). Water is acidified by adding 10 mL glacial acetic acid per 800 mL water;

V Sample filters used are alcohol compatible 0.45 pm nylon 66 membrane.

9.2.5 Microbial contaminant limits

To carry out the analysis for microbial contamination, beans are taken from many different boxes of a shipment of beans, so that a representative sample is obtained. A weighed sample of beans is then suspended in sterile distilled water and shaken on a shaker for at least 1 hour. The beans are then removed and the extraction liquid is diluted serially with sterile distilled water for appropriate microbial load for culture and testing.

To insure the microbial safety, beans are tested for contaminants, as shown in Table 9.4: MPN stands for Most Probable Number and is a standard parameter of reporting E. coli analysis.

Table 9.4 Recommended limits for contaminants

Test | Recommended Limits, Counts/g

Total Plate Count | 1,000-10,000

Yeast | <10

Mold | <1

E. coli 3 Tube MPN | <3/g

Salmonella | Negative

Contaminant levels exceeding the recommended limits indicate improper handling and unhygienic conditions during curing and storage.

9.3 QUALITY CONTROL OF COMMERCIAL VANILLA PRODUCTS

9.3.1 Definition of vanilla products

The US Code of Federal Regulations for Vanilla, 21 CFR 169.175-169.182, defines commercial vanilla products.

9.3.1.1 Vanilla extracts