Info

(4) Pipette approximate amount of dilute solution (depending upon its colour value (CV)) into a 100 ml Nessler tube. Bring up the volume to mark with acetone. Compare through the length of the tube with blank. Make small addition until sample and blank match.

(5) Calculation:

Where A is the volume of dilute solution used (in ml) and B is the percentage of dilute solution.

(6) Blank: Into a 100 ml Nessler tube, pipette 10 ml of 0.1 N potassium dichromate solution (4.904gm K2Cr207 per litre) and 1.0ml of 0.5 N cobaltous chloride solution (5.948gm CoCl2-6H20 per 100ml) and make up to 100ml with distilled water.

by instrumental method

Spectrophotometric assay of oleoresin paprika (Videki and Videki, 1961; Pruthi, 1999):

(1) Equipment:

(i) Spectrophotometer: Beckman Model DU or DK2.

(ii) 1 cm silica or corex capped cell.

(2) Procedure: Weigh l.Og of oleoresin into a 100 ml volumetric flask, and make up the volume with acetone. Pipette 1.0 ml of this solution into a second 100 ml volumetric flask and make up the volume with acetone.

Using a tungsten lamp source and a P28 photomultiplier tube, zero the spectrophotometer using acetone as a blank. Measure the absorbance of the 0.01% solution oleoresin at 458 nm. Multiply this reading by 61,000 to obtain the Nesslerimeter colour value.

Solubility in different solvents

Alcohol: Partly soluble with oily separation.

Benzyl benzoate: Soluble in all proportions.

Fixed oils: Soluble in all proportions in most fixed oils.

Glycerine: Insoluble.

Mineral oil: Very slightly soluble.

Propylene glycol: Insoluble.

Determination of residual solvent in oleoresin

By distillation—gas chromatography method (E0A, 1979)

equipment and chemicals

(1) Gas chromatography

(2) "Volatile oils apparatus" for essential oils heavier than water, made according to the ISU specification (J. Am. Pharma. Soc. XVII (4), 346). The neck on the distilling head should be insulated with asbestos (Pruthi, 1999).

(3) Pipettes, volumetric flasks, heaters, etc.

(4) Toluene of purity suitable for this analysis. Purity of the toluene for the purpose of this analysis may be determined by a gas chromatographic analysis using the columns and conditions prescribed below and injecting the same quantity of toluene as will be injected in the analysis for solvents. If impurities interfering with this test are present in the toluene, they will appear as peaks prior to the toluene and can be removed by fractional distillation.

(5) Column packings: (a) 17% by weight of Ucon 75-H-90,000 on 35-80mesh chromosorb-W or equivalent support, (b) 20% Ucon LB-135 on 35-80 mesh support, (c) 15% Ucon LV-1715 on 60-80 mesh support, (d) Porapak Q 50-60 mesh.

(6) Potassium carbonate (anhydrous), sodium sulfate anhydrous, a detergent free of volatile compounds, and an anti-foam free of volatile compounds. If volatile compounds are present in the detergent or anti-foam, they may be removed by length boiling of their aqueous solution.

(7) Benzene of sufficient purity for this analysis; purity is to be determined as in the case of toluene.

reference solution

(a) Prepare a solution of toluene containing 2,500 ppm of benzene. If the toluene available contains benzene as the only impurity, the benzene level in the toluene can be determined, and sufficient benzene can be added to bring the level to 2,500ppm. A procedure for determining the benzene content of toluene is as follows.

Procedure'. At constant carrier gas flow rate and column temperature, with other instrumental condition constant, determine the area of benzene in a suitable sample size of toluene. Add known amounts of benzene to this toluene, and determine areas for each known amount under the same conditions. Plot the results on a linear graph paper, with the known concentration of benzene as the ordinate and the area as the abscissa. The plot should be linear, and the negative intercept of the ordinate will give the concentration of the benzene in the original toluene.

(b) Prepare a solution containing 0.63% v/w acetone in water.

Procedure-. Place 50.Og of oleoresin, 1.0 ml of solution A, lOg of sodium sulfate, 50 ml of water, and a small amount of detergent and anti-foam in a 250 ml flask. Attach the distilling head and receiver and collect approximately 15 ml of the distillate. Add 15g of potassium carbonate to the distillate, cool while shaking, and allow the two phases to separate. All the solvents except methanol will be present in the toluene layer. Draw off the aqueous layer, and place it in a 50 ml flask with boiling chips. Add 1.0 ml of reference solution B. Attach a distilling head and distill off approximately 1.0 ml. This distillate will contain methanol if present in the oleoresin, and acetone as the internal standard.

Sample size'. 15—20 |xl for hotwire detector.

(1) Ucon 90,000: Use this column for the separation of acetone and methanol from their aqueous solution. Use it for the separation and analysis of hexane, acetone and trichloroethylene in the toluene layer from the distillate. Elution order on this column is acetone, methanol, water; hexane, acetone, isopropanol plus methylene chloride, benzene, trichloroethylene, ethylene dichloride plus toluene.

(2) Ucon LB-135: Use this column for the separation of ethylene chloride, isopropanol and ethylene dichloride. Elution order on this column is: hexane plus acetone, ethylene chloride, isopropanol, benzene, ethylene dichloride, trichloroethylene, toluene.

(3) Ucon LB-1715: This is the best general purpose column, except for the determination of methanol. Elution order for this column is hexane, acetone, benzene, ethylene dichloride, toluene.

computations

(1) Calibration of the instrument. Determine the response of the detector for known ratios of solvents by injecting known mixtures of solvents and benzene in toluene. The levels of the solvents and benzene in toluene should be of the same magnitude as present in the analysis. Calculate the areas of the solvent in relation to benzene. Compute the calibration factor C of the detector as follows:

The recovery of the various solvents from the oleoresins in relation to the recovery of benzene by the distillation procedure is:

hexane 52%; methylene chloride 87.5%; acetone 85%; trichloroethylene 113%;

isopropanol 100%; ethylene dichloride 102%; methanol 87%.

(2) Calculation of residual solvent in oleoresin: The level of the internal standards, related to the 50g of oleoresin used, is: Benzene 43.4 ppm and acetone lOOppm. The level of residual solvent using benzene as an internal standard is computed as follows:

Residual solvent ppm = 43.4 X C (solvent) X 100/(% recovery of solvent X area of solvent/area of benzene. The level of residual methanol using acetone as an internal standard is: Residual methanol ppm = 100 X C (methanol)/0.87 X area of methanol/area of acetone.

By gas chromatography head-space (alternate) method (E0A 1979)

(1) Principle: an oleoresin sample is diluted with diethyl phthalate and equilibrated at 60°C with its head-space in a flask sealed with a septum. A sample of head-space vapour is assayed by gas chromatography. The residual solvent content of the oleoresin can be obtained from that of the vapour by use of a calibration chart.

(2) Apparatus: (a) Bottles, glass, 2oz bottle equipped with septum caps; (b) injection syringe, 2.5 ml, gas tight; (c) Water-bath, set the thermostat at 60°C; (d) Gas chromatograph (GC) with flame ionization.

(b) Packing: Poropak Q (or other suitable column packing)

(c) Column temperature: 150°C, isothermal.

(d) Injection port temperature: 220°C.

(e) Detector: Flame ionization

Hydrogen flow rate: 50ml/min. Air-flow rate: 500ml/min.

(f) Carrier gas: Nitrogen, flow rate : 40ml/min.

wt% solvent wt% benzene x area of benzene area of solvent

(a) Residual solvent standard (RS) essentially single peak by GC.

(b) Diethyl phthalate (DEP) essentially single peak by GC.

(5) Procedure:

(a) Preparation of solutions:

(a-1) Sample solution: Weigh 10 g of the oleoresin to be tested into the 2 oz bottle and make up to 20 g with DEP. Seal with a septum cap. (a-2) Calibration solution: Weigh 1.0 g of RS standard in a 100 ml flask, and make up to 100 g with DEP to obtain a standard solution. Into a series of 2 oz bottles, weigh 1.0, 2.0, 3.0, 4.0 and 5.0g of standard solution and make up to 20 g with DEP. Seal the bottles with septum caps. These calibration solutions will contain respectively, 10, 20, 30, 40 and 50ppm of RS.

(b) Calibration and determination:

(b-1) Immerse the bottles up to the necks in a water-bath thermostated at 60°C. After 15 min remove the bottles, mix by whirling, and return to the water-bath for a further period of 2 h. (b-2) Using the 2.5 ml syringe, inject a sample of the vapour in each bottle onto the GC column, as follows: draw 2.5 ml of air into the syringe and insert the needle into the bottle. Discharge the syringe contents into the bottle and withdraw 2.5 ml of vapour from the bottle. Discharge the syringe contents back into the bottle, again withdraw, and repeat these operations once more. Leave the needle in the bottle for 30 s, withdraw the needle, and inject the syringe contents into the GC column.

(c) Calculation:

(c-1) Determine the areas of the RS peaks from the gas chromatograms of the calibration samples and plot in a graph the corresponding concentration of the RS. A linear calibration curve passing through the origin should be obtained. (c-2) Determine the area of the RS peak from the gas chromatogram of the oleoresin sample and read from the calibration curve the concentration of RS expressed in ppm, correcting, if necessary for attenuation.

Quality of oleoresin Capsicum

The oleoresin Capsicum is obtained by solvent extraction of powdered dried ripe fruit of

Capsicum frutescens (bird chillies). Stahl (1965) has presented a critical review of the available methods of quality assessment of oleoresins.

Appearance and odour

A clear red, light amber or dark red, somewhat viscid liquid with a characteristic odour and very high bite.

Scoville units

The method is the same as given for chilli oleoresin (min Scoville units 240,000).

Odour value

Four thousand units maximum. The paprika method is the same as for paprika oleoresin.

Residual solvent As described here:

(a)

Alcohol:

Partly soluble with oily separation and/or sediment.

(b)

Benzyl benzaoate:

Soluble in all proportions.

(c)

Fixed oil:

Soluble in all proportions in most fixed oils.

(d)

Glycerine:

Insoluble.

(e)

Mineral oil:

Insoluble.

(f)

Propylene glycol:

Insoluble.

Quality of oleoresin red pepper

The oleoresin red pepper is obtained from Capsicum annuum L. or its hybrid pepper, known as "Louisiana sport".

Appearance and odour

A deep red coloured liquid with a characteristic odour and high bite.

Scoville heat units 24,000 max.

Residual solvent: As described here:

(a)

Alcohol:

Partly soluble with oil, separation or sediment

(b)

Benzyl benzoate:

Soluble in all proportions.

(c)

Fixed oils:

Soluble in all proportions in most fixed oils.

(d)

Glycerine:

Insoluble.

(e)

Propylene Glycol:

Insoluble.

Quality of oleoresin chilli

Oleoresin chilli is extracted from chillies (the fruit of red pepper, Capsicum annuum L. or Capsicum frutescens L.) using approved food grade solvent and subsequent careful removal of the residual solvent by distillation. In addition to the intense pungency caused by the capsaicin and the small quantities of allied alkalloids, the oleoresin chilli has dark red carotenoid pigments. The oleoresin should be free from rancid flavour.

Determination of the colour value of oleoresin chilli

(a) Principle: To make the estimation completely objective, instrumental analysis is employed. The colour of a specified dilution is estimated at 458 nm and multiplied by the appropriate factor (ISI, 1979; ISO, 1989).

(b) Apparatus'. (1) 100 ml volumetric flask; (2) 1ml pipette; (3) Spectrophotometer or a Spectronic photo-electric colorimeter with adjustable wavelength.

(c) Reagent: Acetone.

(d) Procedure: Weigh l.Og of oleoresin into a 100 ml volumetric flask and make up the volume with acetone. Using a tungsten lamp source and acetone as the blank, measure the absorbance level of the 0.01% solution of oleoresin at 458 nm. Multiply this reading by 61,000 to obtain the colour value.

Determination of Scoville heat units in oleoresin chilli

(a) Principle: This is a method based on sensory evaluation. Although subjective, industry and trade still employ this method. Oleoresin solutions variably diluted with sugar solution are tested in increasing concentration. The highest dilution at which a pungency is first detected is taken as the measure of the heat value (ISO, 1977; ISI, 1976).

(b) Reagents'. 5% sugar solution 95% alcohol.

(c) Procedure:

(a) 0.2 g of the oleoresin is weighed into a 50 ml volumetric flask. 95% alcohol is added up to the volume, the material is shaken and allowed to settle.

(b) To 140 ml of 5% sugar solution, add 0.15 ml of alcoholic solution. Swallow 5 ml of this solution after thoroughly shaking it; and

(c) A bite or stinging sensation in the throat which is just perceptible at this dilution is equal to 240,000 Scoville units. If bite is very strong, a further dilution is necessary. The following dilutions are equal to the corresponding Scoville heat units when 5 ml of swallowed causes a perceptible pungent sensation:

(i) 15 ml of sugar solution, 30 ml of solution (b) = 360,000

(ii) 20 ml of sugar solution, 20 ml of solution (b) = 480,000

(iii) 30ml of sugar solution, 15 ml solution (b) = 720,000.

A panel of five members may be used. Three out of the five members should find the solution to be pungent.

Determination of capsaicin content

The pungency of chilli is caused by capsaicin and traces of allied pigments. Therefore, pungency can be more objectively evaluated by estimating the capsaicin content. For this purpose, two methods are currently used and are equally suitable (Govindarajan et al., 1977; ISI, 1976; ISO, 1988).

tlc method for capsaicin determination

(a) Principle: The capsaicin and allied pigments may be separated from other constituents by TLC on a silica gel. The pungent principle can react with Folin-Dennis reagent to give a blue complex which may be estimated colorimetrically. The HPLC and spectrometric methods have been described earlier for the determination of capsaicin content of chillies (whole and ground). The presence of much simpler TLC/PPC methods are briefly described below:

(b) Apparatus: (1) Complete TLC set; (2) Spectrophotometer or colorimeter capable of measuring in the region of 660—669 nm; (3) 10 ml measuring jar with stopper; (4) Test-tubes; (5) Graduated 10 ml pipettes (graduated at 0.1 ml).

(2) Developing solvent — Mixture of 80 ml of benzene and 5 ml of methanol;

(3) Phosphomolybdic—Phosphotungstic acid (Folin-Dennis Reagent)-Reflux for 2 h, 750 ml water, 100 g sodium fungstate, 20 g phosphomolybdic acid and 50 ml of phosphoric acid. Cool the mixture and dilute to 1 L with water.

(4) Vanillin - Analytical grade.

(5) Standard aqueous vanillin solution — The standard aqueous solution of vanillin should contain 0.01 mg/ml. This standard solution (1—3.5 ml) is made to react with 0.5 ml of

Folin-Dennis reagent and 1 ml of saturated aqueous solution of sodium carbonate, keeping the total volume to 5 ml by the addition of distilled water.

(d) Procedure:

(1) Preparation of TLC plates: Mix 30 g of silica gel containing calcium sulphate as a binder. Mix with 60 ml of distilled water. Pour the slurry into a TLC spreader adjusted to a thickness of 150 |xm and spread over five to six glass plates of 20 X 20 cm. Air dry the plates for 4—5 h, and later activate them by drying them in a desiccator.

(2) Estimation: Weigh 5 g of oleoresin in to a 10 ml stoppered measuring jar and make up the volume with acetone. Spot 10 |jl1 (5-20 (jl! depending upon the capsaicin content) of this solution on TLC plate and pour developing solvent into an all glass chamber which has been thoroughly saturated with the vapours of the solvent. Expose the plate for about half an hour to free it of the solvent and lightly spray with phosphomolyb-dic-phosphotungstic reagent. Mark the clear blue capsaicin spot (Rf 0.16) with a small stainless steel scooper or spatula in the form of a circle, enclosing both the spot and an area of 0.25 cm beyond the spot. Scoop out the silica gel containing capsaicin in the marked area into a clean butter paper and transfer it into a test-tube. Add 3.5 ml of distilled water and shake it well. Pipette out 0.5 ml of phosphomolybdic—phospho-tungstic reagent into the tube and mix well. After 3 min add 1 ml of a saturated aqueous sodium carbonate solution. Mix thoroughly for 5 min and set in aside for 1 h. Prepare a reagent blank using silica gel layers from a blank area in the plate. Centrifuge the tubes (or filter) at the end of one hour to separate solids. Read optical density in a spectrophotometer at 725 nm (or in a colorimeter using a glass filter in 660—690 nm region). Determine the amount of capsaicin (X) in the spot from a standard graph of known concentrations of pure vanillin vs optical density. (Multiply by a factor of 2 to correct for the difference in the molecular mass of capsaicin and vanillin.)

(3) Preparation of standard graph: Different volumes of a standard aqueous solution of vanillin are made to react with Folin-Dennis reagent and the optical density is determined. Draw a standard graph with the mass of vanillin against optical density (Govindarajan and Ananthakrishna, 1970).

(4) Calculation:

Capsaicin (%) = X X 1000 X 200/r where X is the number of grams of capsaicin (vanillin X 2) in the spot, and r is the number of (jlI of 50% acetone solution of oleoresin.

(b) paper chromatographic method for capsaicin determination

(a) Apparatus:

(1) Chamber for ascending paper strip chromatography, complete with accessories.

(2) Spectrophotometer or colorimeter with narrow band filter.

(b) Reagents'. (1) Ethyl acetate solvent of capsaicin or dilution of total extracts; (2) Methanol (AR); (3) Boric acid (AR); (4) Potassium chloride; (5) Buffer-A solution containing 3.1 g of boric acid and 3.7 g of potassium chloride in 100 ml of distilled water adjusted to pH 9.6 with 1 N sodium hydroxide; (6) Methanol Buffer pH 9-6 (60 : 40 v/v) and developing solvent; (7) Gibba reagent 0.1% 2,6-dicholro-/;-benzoquinone-4-chloromine in acetone as the chromogenic reagent.

(1) Dissolve the oleoresin or extractives in ethyl acetate to give a solution containing about 2.5 g/L of capsaicin. Apply an amount of the solution containing 10-50 (Jtg of capsaicin (that is 4—20 (jlI) as a thin streak covering the entire width of a 2 X 20 cm strip of Whatman no. 3 paper. Develop by ascending chromatography in methanol buffer solvent until the solvent front has moved up about 15 cm, usually in about 1 h. Dry the strip in air (Govindarajan and Ananthakrishna, 1974, ISI, 1979).

(2) Pass the dried paper strip uniformly through Gibbs reagent and dry it in air. Capsaicin becomes faintly visible as a blue spot near the solvent front. Develop the colour further by spraying it with buffer (pH 9-6) lightly on both sides of the paper. Transfer the strip to a dark cupboard for 30min for the full development of colour and drying.

(3) Excise the blue spot and elute it with methanol in the dark. Make up the eluted colour to a convenient volume (5—10 ml) and measure at 615 nm in a spectrophotometer against a reagent blank eluted from the corresponding area of strip run simultaneously without the sample. Calculate the percentage of capsaicin from a standard graph as given in Section "TLC method for Capsaicum Determinants" or by using E 1% cm = 640 at 615 nm.

(d) Estimation of capsaicin content in Capsicum: Prepare the oleoresin or total extract of the sample by Soxhlet extraction of the powdered Capsicum with a suitable solvent (ethyl acetate) for 2.5—3 h. After removal of the solvent, weigh the residue and proceed as in "TLC method for Capsaicum Determinants".

(e) Standard curve and E 1%/1 cm for pure capsaicin: Prepare a solution of pure capsaicin in ethyl acetate (2 g/L). Spot 10, 20, 30, 40 and 50 (Jtg of capsaicin (5-25 Ul) on strips of Whatman no. 3 paper. Develop and visualize the capsaicin spot elute it, and proceed further, as usual, for the estimation of capsaicin.

Development of International (ISO) Standards for spices including chillies and their methods of testing

Organizational set up and objectives of International Organization for Standardization

The International Organization for Standardization (ISO) is the specialized international agency for the development of international standards. The ISO has 191 technical committees covering different commodities traded on an international level. The membership of the ISO, at present, comprises 89 countries. The ISO aims to promote the development of standardization with a view to facilitate the international exchange of goods and services and to develop cooperation in the sphere of intellectual, scientific, technological and economic activities. The results of the ISO technical work are published as international standards (ISO standard).

One of the ISO technical committee, ISO/TC 34, deals with agricultural food products. This technical committee has 15 sub-committees dealing with various food commodities, such as fruits and vegetables, milk and milk products, meat and meat products, spices, etc. One of the sub-committees (ISO/TC 34/SC 7) deals with spices and condiments. The Indian Standards Institution (now called the Bureau of Indian Standards, BIS), New Delhi, holds the ISO/TC

Table 3-11 International (ISO) standards for Capsicums, chillies, paprika products and methods of testing

ISO standard no. Title of ISO specification Total pages

ISO standards for whole and ground Capsicums, etc:

Chillies and Capsicums, whole or ground (powdered) — specification Ground (powdered) paprika (Capsicum annuum L.) — Specification 4 Determination of degree of fineness of grinding — hand seving 2

method (reference method) Ground (powdered) paprika (Capsicum annuum L.) — 8

Microscopical examination Chillies - determination of Scoville index

Ground (powdered) paprika — determination of total natural 3

colouring matter content Chillies and chilli oleoresins — determination of total capsaicinoid content, Part 1: Spectrometric method Chillies and chilli oleoresins - determination of total capsaicinoid content, Part 2: Method using High Performance Liquid Chromatography (HPLC method)

International (ISO) methods of test spices and spice-products:

ISO 2825: 1981 Preparation of a ground sample for analysis

ISO 948: 1980 Method of sampling

ISO 927: 1982 Determination of extraneous matter

ISO 1298: 1982 Determination of moisture content — entrainment method

ISO 941: 1980 Determination of cold water-soluble extract

ISO 6571: 1984 Determination of volatile oil content

ISO 928: 1997 Determination of total ash

ISO 1108: 1992 Determination of non-volatile ether extract

ISO 930: 1997 Determination of acid-insoluble ash

Source: Compiled by Pruthi (1999)-

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