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I <ULh*JU

LIBRARY

I S . IAL I ECORD

NOV 2 J 1957 *

U. S. DEPARTMENT OF AGRICULTURE

IiHTHODS Ui»LD AT IfiiSTaitti thGIO^Al jtL3h.AKCH -UbO.^.TOiff FOR LXTRACTIOfJ AND ANALYSIS OF P£CTIC HA Tji RIALS x

H. 5.v_Qwens, R. ii. (^cCready, A. D, I Shepherd, T. H. techultz, lie L0 IPippen, H. A. \Swenson, J. C. U.iers, R. F. (^rlandsen, and1./. D. ^..aclay

Western Regional Research Laboratory 3 Albany, California

Kethods used in the Western ;topional Research Laboratory for the extraction, isolation, purification, and analysis of pectic substances are described in this publication,. Procedures are presented for the extraction of total pectic substances and for the extraction, isolation, and purification of undegraded pectin for characterization. Specific directions are £iven for the determination of ammonia, moisture, ash, equivalent weight, rcethoxyl, anhy- drouronic acid, acetyl, viscosity, jelly grade, setting time, and specific optical rotation, methods of assay for pectinesterase (PiL) and polygalacturonase (?G) are in- cluded. Later napes contain references and a list of cur- rent nomenclature of pectic substances and enzymes*

UNITED STATES DaPANTilENT OF AG ►ilCULTU -iL

CONTENTS

Page

EXTRACTION 1

Total Pectic Substances 3

"Water-Soluble h Pectin k

Cold- Extracted Pectin I4.

Isolation and Purification $

QUALITATIVE TESTS $

Pectic Substances f>

Ammonia 6

QUANTITATIVE ANALYSIS 6

Ammonia 6

Moisture 6

Ash 6

Equivalent Weight 7

Methoxyl Content 7

Anhydrouronic Acid 8

Acetyl 9

Viscosity 10

Number Average Molecular Weight 12

Jelly Grade ll±

Setting Time 17

Optical Rotation 17

PECTIC ENZYMES 18

Pectinesterase (Pectase, Pectin Methylesterase) 18

Polygalacturonase (PG) 18

Other Pectic Enzymes 19

LITERATURE CITED 1?

NOMENCLATURE 2h

Methods for Extraction and Analysis of Pectic Materials Used at Western Regional Research Laboratory

H. S. Owens, R. M. McCroady, A. D. Shepherd, T. H. Schultz, E. L. Pippen, H. A. Swenson, J. C. Miers, R. F. Erlandsen, and W. L. Maclay^

Pectic substances are almost universally distributed in plant tissue, where they act as cementing materials between cells, particularly in fruit tissue.!/ Their importance in food technology can hardly be overemphasized, because they play important roles in the maintenance of texture in fresh fruits and vegetables and also in the viscosity or gel strength of processed foods like catsups, chili sauces, jellies, preserves, and low-solids gels. Problems involving pectins are continually arising. Accordingly information on the measurement, composition, and properties of pectin is essential.

Formation of jellies is impossible without pectin, and knowledge of its amount and jelly grade in the fruit juice to be, gelled aids in estimation of amounts of pectin or pectin concentrate to be added. £/ The setting time of pectin controls settling of solid particles in marmalades and jams. In fruit purees and tomato products which must have a high viscosity, the amount and properties of the dissolved pectin are important factors, but knowledge of these factors alone will not enable one to solve all the problems in this field. For example, the shepe and content of fiber in purees materially contribute to their flow behavior. Pectin and other water- soluble polysaccharides on the surfaces of those fibers probably contribute to the flow properties, but this contribution has not been eve lusted.

Increase of quantitative information on pectin in foods will resolve many problems in food technology. The purpose here is to provide more specific directions on methods used in this Laboratory for extraction, analysis, and evaluation of pectin, and to provide more information on analytical and physical methods than is avail- able in the publications by Berglund (3), Hirst and Jones (19), Henglein (16), Pallnann and Deuel (44) , Joslyn and Phaff (24), and Kertesz"T26 ) . Tho methods de- scribed here are intended to provide background information concerning pectic sub- stances. Although these methods have practical value in problems related to tex- ture and consistency, their use may also indicate a new approach to such problems. This field of endeavor, as it applies to woody tissue and to raw material used as a source of pectin, is under investigation by a subcommittee on pectin in the Cellulose Division of the American. Chemical Society.

Extraction

It hes been suggested that Ma pectin determination is somewhat empirical and has in it not a littlo art and philosophy." An unresolved question remains whether pectic matorials are present in plant tissue as protopectin and as pectate (4) or as a high-molecular-weight pectin cross-linked through cotions or hydrogen bonds, or physically entrappod (15, 43, 47). Until the question receives a definite answer, "philosophy" will enter tEe decisions on best extraction procedures. We have assumed that pectin is present primarily as a high polymer, with a wide

l/ Nomenclature of pectic substances is listed with definitions on page 2k>

2/ The term "jelly" refers to gel systems containing not less than 65 percent soluble solids. See regulations of Food, Drug, and Cosmetic Administration.

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distribut ion of molecular weights and some variation in methoxyl content. Except for salt linkages, no primary bonds between pectin and cellulose or other high polymer are assumed to exist. The simplest and least drastic extraction methods based on these assumptions have been chosen to obtain sufficient information to characterize pectic substances in various fruits and vegetables.

Evidence of intimate association of pectic substances with cellulose and other poly- saccharides in cell walls is convincing and permits deductions concerning con- ditions for their extraction. First, pectin is macromoiecular ; therefore plant tissues from which it is to be extracted must be maintained in a swollen condition to allow its diffusion. Second, the presence of carboxyl groups in pectin offers opportunities for cross linking with other pectin molecules, proteins, and gluc- uronic acid groups in cellulose through polyvalent cations. Heat, acid, or cation-acceptors, or some combination of these agents, is required to disrupt these cross linkages. Third, hydrogen bridges between associated high polymers and salt linkages with proteins can be dissociated by thermal energy and addition of suit- able hydrogen-bond-breaking agents.

Vnater was chosen as part of the extraction medium because it is s satisfactory solvent for both high-methoxyl pectins and many salts of low-methoxyl pectins and pectates. Of the extraction aids, three have been given the most attention. Acid can be used for tissue containing only small amounts cf pectates; otherwise it is unsatisfactory. It is the recommended agent if information is desired on the com- mercial possibilities of plant tissue for jelly-type pectin, ^odium polymetaphos- phate (CaLgon)^/ and sodium salt of ethylene diamine tetra-acetic acid (Vorsone, Regular) V are the only cation acceptors considered. They form soluble complexes with polyvalent cations. Calgon, however, is difficult to remove from alcohol precipitates of pectin. Versene must be used at a relatively high pH at which pectin may be degraded. We have no reason to believe that pectins from all plant sources are the same; therefore pH stability of pectins from different plants should be checked before pH values of 6 or higher are used for the cold extractions. For total pectic substances this effect is net serious. A hot extraction method has been chosen for total pectin becauso of its effect on hydrogen bridges and the higher rate of diffusivity of pectin in hot solutions.

The nature of the problem and the nature and extent of information that must be obtained about the pectin in situ will determine how drastic the extraction pro- cedure can be. For estimation of total pectin, extraction conditions will gener- ally be more drastic than those required for removal of a pectin that has the molecular weight and degree of e sterif icat ion of that normally present in the tissue A number of methods have been developed to obtain maximum amounts of information about pectin in plants. Kertesz and McColloch (27) use successive extractions with water, oxalate, and acid solutions. Their results indicate that tomato pectins extracted by water and oxalate differ in degree of esterification. In apples (13) water-soluble and Calgon-extractable pectinates have about the same degree of esterification. A complete reviow of methods will not be given here, but only those methods will be presented that we generally use and consider most satis- factory from the present inconclusive knowledge of pectic substances.

3/ Mention of these products and others by commercial name does not constitute ~~ recommendation by the Department of Agriculture over other products not mentioned .

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The methods recommended cover determination of total pectic substances and ex- traction of pectic substances in the cold with a metal-ion sequesterant to obtain materials for characterization. A procedure for determination of water-soluble pectin is given for its interest in cortain fields. The minimum information that should be obtained on the cold-extracted, carefully precipitated, and deashed material includes percent anhydrouronic acid, percent methoxyl, percent acetyl, and intrinsic viscosity. With knowledge of amount of pectin and characteristics of cold-ext ractable pectin in the original and in processed tissue, conclusions can be drawn concerning the presence of pectic enzymes or whether the processing treatment was excessively drastic. Such conclusions can be drawn if the amount of material extracted in the cold is sufficient to make it representative of all the pectic substances. Only experience will enable one to set a limit for the amount of material that is typical.

Total Pectic Substances; Y/hen it is necessary to know the total amount of pectin (for example during storage tests of foods, studies on composition of foods, or estimation of recovery in commercial production of pectin) rather drastic ex- traction conditions are required. The method below yields results between 90 and 100 percent of those obtained by direct anhydrouronic acid analysis of grapefruit, cranberries, raspberries, and carrots and between 95 and 100 percent of results obtained by R. M. McCready and Elizabeth A. McComb of this Laboratory, whose method, unpublished at present, is more specific and involves extraction with Pectinol. W

Enzymes, if present, can be inactivated by treating the tissue with boiling alcohol as described on page 1| . If the tissue is dried, freeze-drying or other methods which maintain a porous structure should be used, or the dried material should be ground to pass a 60- mesh or smaller screen.

Weigh 100 gm. of fresh or 10 gm. of ground dried tissue into 400 ml. of water. Macerate fresh tissue thoroughly in a blender. Place the slurry in a tared container; add 1.2 gm. of freshly ground Calgon; adjust to pH 4.5, and heat slowly to about 95° C. while stirring. 4/ Continue stirring and heating at 90-95° for 60 minutes. It is advis- able to check the pH after 15 minutes to be sure it is at 4.5. At the same time water lost by evaporation is replaced. Do not add water during the last 20 min. of the extraction period. Add 4 gm. of filter aid and 4 gm. of ground paper pulp. Check the weight. Filter rapidly through an 11-cm. filter, using a fast filter paper coated with 3 gm. of moistened fast filter aid. Collect not less than 200 ml. Cool the filtrate as rapidly as possible.

The cooled filtrate is analyzed for its pectic content by the optical rotation, the CO2 determination, or the carbazole method, as described later. In case re- agents and equipment are lacking for these methods, the pectic substances are pre- cipitated by alcohol as described on page 5. In any case results should be

4/ Alternatively , the slurry is adjusted to pH 6 after adding 2 gm. of Versene. A pH of 1.8 to 2.0 without sequesterant can also bo used with most tissue but is not effective when low-methoxyl pectin or pectate is present.

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calculated to include the pectic substances held up in the filter pulp as well as that in the filtrate. For example, if the extract contained 300 gm. of water immediately prior to filtration (including that in the tissue) while the filtrate contained only 200 gm. of water, the amount of pectic substances in the filtrate would be multiplied by the ratio 300/200 to calculate the amount in the original tissue. This calculation saves two or more extractions.

These directions are satisfactory if the raw material is as rich in pectin as citrus peel (3 percent). Most sources require a larger sample and a lower ratio of water to sample. With fruits a 2-te-l ratio will be better than the 4-to-l given above. If the carbazole procedure is used for determination of the uronide content, all quantities can be greatly reduced so that operation can be carried out in centrifugo tubes.

Pectin obtained by this method will be somewhat degraded but is satisfactory for laboratory exercises on the properties of pectin. Its properties are a rough indication of the care with which the source material was treated.

^Water-Soluble" Pectin: Problems concerning viscosity of purees, tomato products, and jellies require information on the amount and nature of pectin liberated during processing and some knowledge of fiber content and cations present in the medium. Usually addition of 1 percent of filter aid and 1 percent of ground paper pulp will permit pressing or filtering the juice. Isolation of the pectin present is accomplished by directions given on page f> • Yields of this material are not significant unless particle size is controlled,

Cold-Extracted Poctin: To supplement information available from the amount and nature of the so-calleduwater-soluble" pectin, it is desirable to isolate pectin which is comparable to that in situ. Pectic enzymes must to inactivated. This is accomplished by slicing the tissue into 95 percent ethanol above 70°C. ^he final ethanol concentration should be not less than 70 percent. The heating period should be about 12 to 18 minutes. To maintain a porous structure and to prevent possible loss of pectin by reaction with other compounds, it is advisable to store the alcohol-treated tissue either under alcohol or in the frozen state. If it is dried, a freeze-drying or other vacuum drying technique should be used. It will be noted that methods of pretreating the sample to obtain a pectin representative of that in situ are much less drastic than those for total pectin. We have found that prolonged heat treating and drying of samples at high temper- atures are unsatisfactory if a high-mclecular-weight pectin is desired. The pro- cedure follows:

Blend 100 gm. of tissue (previously treated with alcohol) with 200 gm. of water. Add 2 gm. of Calgon or Versone. Adjust the pH to 4.5 for the phosphate or 6 for the amine derivative. After 24 hours at room temperature, with occasional stirring, add 3 gm. of filter aid and 3 gm. of paper pulp, heat to 60°c., and separate the solution by filtration, centrif uging , or pressing. Accomplish isolation as de- scribed under that section except that 1 volume, or between 1 and 2 volumes, of alcohol or acetone containing acid are used for the pre- cipitation. Two precipitations are recommended, followed by washes with neutral 70 percent solvent and finally pure solvent. If it is desired to remove 80 percent or more of the pectin, a second extrac- tion is recommended. Toluene or other antiseptic should be used during these extractions.

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Cold-extracted pectin is prepared primarily to study properties of a pectir. that is comparable to that in situ* It should bo carefully de-ashed, freed of excess acid, and dried at no more than 60°C. in vacuo.

Comparisons between cold-extracted and water-soluble pectin should be made in a sufficient number of cases to determine whether or not they are the same. Pectin is located in and between the cell walls and possibly is out of contact with pectic enzymes until the cells are ruptured. The pectin in the juice might bo the result of leaching of the membranes near cell ruptures and thus would be typical of in situ pectin. Cold sequesterants decrease the number of salt link- ages involving pectin, facilitating removal of an undegraded pectin.

Isolation and Purification; Pour the cooled, weighed, or measured filtrate into 3 volumes of ethanol, 2-propanol, or acetone containing 0.5M concentration of HC1. (The pH of the slurry should be between 0.7 and 1.0.) Stir for one-half hour. Centrifuge, filter, or separate the precipitate on coarse-mesh nylon (bolting cloth). A second wash at the same pH is recommended to remove all but traces of ash. Wash repeatedly in 400-ml. portions of 70 percent alcohol or acetone until tho precipitate is essentially chloride ion-free or the pH is above 4. Dehydrate the precipitate further in 400 ml. of acetone. Dry overnight in vacuo (5 mm. Hg pressure) with a slow stream of dry air passing through the oven. Weigh the precipitate. Determination of methoxyl, ash, and uronido content will yield information on the total amount of pectin. Until more is known about tho composition of pectin, the uronido content will be considered a measure of the total yield of pectin. Pectin yields based on tho alcohol precipitate will be higher by 15 to 50 percent than the uronido values.

If acid is not used in the organic procipitant, the correction for ash will not be reliable (see Ash Determination).

Acetone is used in tho final step before drying to remove any alcohol that might be sorbed by the precipitate, since such alcohol would interfere with Zeisol de- termination for methoxyl (20).

If the concentration rf pectin is below 0.2 percent in tho filtrate, it should be concentrated to that level in vacuo before precipitation in alcohol.

Qualitative Tests

Pectic Substances: A useful qualitative test for the presence of pectic sub- stances is a modification of Ehrlich's lead acetate test (14).

Dissolve or suspend the material under question in water at approx- imately 1 percent concentration of substance to be tested and add 0.2 percent of potassium oxalate and 0.1 percent of Pectinol. Mix and cover with toluene and allow it to stand for 20 or more hours. Remove the solids by filtration or centr if ugat ion and test the solution for galacturonic acid. Add saturated basic lead acetate solution to 5 ml. of solution. If galacturonic acid is present, a white precipitate

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forms and re-dissolves upon addition of excess or an equal volume of the lead reagent. Boil the mixture. Formation of a red precipitate is a positive test for galacturonic acid and therefore of pectin in the original substance.

Ammonia: The presence of ammonia will interfere with many of the proposed analy- tical methods.

Add 1 ml. of 0.1N NaOH to a small amount of dried sample. On heating, the presence of ammonia can be detected by its odor or, better, by moistened litmus paper. Wash out ammonium ion with acidified 60 per- cent alcohol, followed by neutral alcohol to remove acid.

Quantitative Analysis

Pectin samples prepared by the foregoing procedure will be dry and should be stored under dry, cool conditions. In moist climates, however, weighing dry pectin samples is difficult because they rapidly adsorb water vapor. Under these conditions samples should be exposed to the air of the laboratory for 1 cr 2 days until they reach an equilibrium moisture level. The moisture content is then de- termined and a correction made for it on all other analyses and physical measure- ments. Results of analyses and physical measurements should be expressed on a moisture-, ammonia-, and ash-free basis.

Ash-free low-methoxyl pectins and pectic acid are only slightly soluble in water. To prepare solutions of them it is necessary to exercise care in adding dilute alkali to the rapidly stirred suspension to avoid degradation or deeste'rif ication Such samples should dissolve when the pH has been increased to 4 or 4.5.

Commercial pectins are likely to contain reducing sugars which can be removed by washing with 60 percent alcohol. The final wash should be with acetone.

Ammonia : Ammonia is determined quantitatively in pectic substances by distillation from magnesium oxide suspension at pH 10.9. Total nitrogen is determined quantitatively by the conventional Kjoldahl procedure.

Moisture: Weigh 1 gm. of sample ground to pass 80-mesh into a tared moisture dish (metal, 2 in. in diameter with cover). Dry in vacuo (5 to 20 mm. cf Hg) for 4 hours at 100°C. Place in a desiccator and cool to room temperature and weigh. Desiccant should be phosphorus pentoxide cr similar effective agent. The pectin is degraded by the process and should not be used for subsequent measurements. If the sample is to be used for other measurements, drying should be done at 70° C. for 16 hours. One percent should be added to the percent mois- ture to obtain agreement with the Fischer method (22).

Ash: Weigh 1 to 2 gm. of pectic substance ground to pass 80-mesh into a tared crucible. Ignite slowly, then heat 3 or 4 hours at 600°C. Cool the crucible to room temperature in a desiccator and weigh. De- termine the alkalinity of the ash as follows. Dissolve the ash in an excess of 0.1N HC1, 25.00 ml. usually being sufficient. Heat gently

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to boiling and after cooling, titrate with 0.1N NaOH, using phonol- t ha loin as an indicator. Calculate the basicity in terms of carbonate and express the results as ash minus carbonate.

A number of errors are likely to occur in the determination of ash. Volatile im- purities such as ammonium salts will give low results. Some cations, notably aluminum, iron, and others, form insoluble oxides which will not be titrated in the determination. It is bettor to de-ash the pectin with acidified alcohol or ion exchange materials as previously described, so that little or no correction for ash is needed.

Equivalent Weight: Values for equivalent weight are used in the calculations for anhydreuronic acid content and degree of ester if ication.

Weigh 0.5 gm. of pectic substance into a 250-ml. titration flask and moisten with 5 ml. of ethanol. One gram of NaCl may be added to sharpen the end point. Add 100 ml. of carbon dioxide-free distilled water and 6 drops of phenol red indicator (or Hinton's indicator (17)-- bromothymol blue 0.4 percent 1 volume, phenol red 0.4 percent 3 vol- umes, cresol red 0.4 percent 1 volume, and distilled water 1 volume). Slowly titrate to avoid possible deesttrif ication, using 0.1N NaOH until the indicator changes (pH 7.5). Be sure all of the pectic sub- stance has dissolved and that no lumping on the sides of the flask occurs. The endpoint should persist for 30 seconds. Save the neutralized solution for methoxyl determination.

1000 x wt. of sample (gm. ) B** wt' I x vol. of alkali (ml ."J

Ammonia- and ash-free pectinic or pectic acids should be used for this determina- tion. Corrections can be made for such constituents, but the results are usually not completely reliable.

Methoxyl Content: The methoxyl content or degree of esterif ication is an im- portant factor in controlling the setting time of pectins, the sensitivity to polyvalent cations, and their usefulness in low-solids gels, films, end fibers.

To the neutral solution titrated for equivalent weight containing 0-5 gm. of pectic substance, add 25 ml. of 0.25N NaOH, shake thoroughly, and allow to stand 30 minutes at room temperature in a stoppered flask. Add 25 ml. of 0.25N HC1 (or an amount equivalent to the base added) and titrate with 0.1N NaOH to the same endpoint as before (17_). ~

N x vol. of alkali (ml.) x 3.1

% MoO = ~ t r

wt. of sample (gm. J

Tho Zeisel method fcr alkoxyl as modified by Shriner (49) offers a precise means of determining the methoxyl content and uses small amounts of sample. Sorbed methanol, ethanol, or 2-propanol must be washed out with acetone or removed by humidif ication. Presence of acetyl does not interfere, but glycosidic and ether methoxyl groups are measured in the same manner as ester methoxyl. The procedure follows :

t

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Weigh 15 to 50 mg. of poetic substance (depending on the methoxyl content) into a small glass container. Place the sample plus a boil- ing chip in the reaction flask and add 2 ml. of melted phenol and 5 ml. of 48 percent HI. Connect tho flask to the remainder of the apparatus, -which consists of an air condenser, a scrubber, end a re- ceiver, Tho scrubber contains 5 percent solutions in equal amounts of sodium thiosulfate and cadmium sulfate, The receivers contain 10 ml. of acetic acid, 1 gm. of potassium acetate, and 15 drops of bromine. Bubble a slow stream of carbon dioxide through the reaction flask end boil the liquid gently with a micro burner. The vapors should con- dense within 8 cm* from the bend of the air condenser. After 30 minutes, wash the contents cf the receivers into a 300-ml. flask with 100 to ,150 ml. of water. Add 1 to 1,5 gm. of sodium acetate hydrate and after solution is complete add formic acid dropwise while shaking the flask to destroy excess bromine. After the bromine is destroyed add 1 gm. of KI , 10 ml. of concentrated HC1, and titrate the liberated iodine with 0»1H sodium thiosulfate using starch as an indicator. The calculation is made from the following:

517 x K x vol. of MzH°Z /a llleO - Q£ 3amp]e (rugTJ

Yfhen the purity of a pectin sample is to be calculated, reduce the methoxyl (-OCHg) to methylene (-CHg), since the anhydrouronic acid analyses include the weight of this oxygen atom in the formula weight of 176.

Anhydrouronic Acid: Although pectin is sometimes considered a partly esterifiod polygalacturonase 5 s it actually contains 10 percent or more of unaccounted- for organic material, which is composed of arabinose, galactose, and perhaps other sugars. Information on the percentage cf anhydrouronic acid is necessary to establish purity and degree of esterif icat ion and to evaluate the physical properties .

If the equivalent weight, methoxyl content, and alkalinity of the ash have been determined the calculation is as follows:

wt . of sample (mg..)

2 ~ m.e. alkali for free acid + m.e. alkali for saponification + m.e. titratable

ash

176 x 100 " • % A.U.A. = 2

A second method for determining the anhydrouronic acid content, which gives the same results as titration, is a modification (35) cf the Lof evre-Tollens (29) method. Carbonates interfere but ash and ammonia do not, because this method de- pends on decarboxylation of the uronide. The procedure follows:

Weigh about 250 mg. of pectic substance into the 125-ml. reaction flask and add 30 ml. of 19 percent HC1. Sweep the apparatus with carbon-dioxide-f reo air and then attach the absorption tower. Add 25 ml. of 0.25N NaOH and a few drops of n-butanol to the absorption tower. Immerse the reaction flask in an oil bath and raise the temperature of the bath to 145° C. while sweeping the apparatus with a stream of

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carbon-diexide-free air for a heating period of 1.5 to 2 hours. Dis- connect the absorption tower, wash the contents into a 300-ml. flask, and add 10 ml. of 10 percent barium chloride solution. Titrate the excess alkali, using 0.1N HC1 until just colorless to phenolphthalein. The value so obtained, expressed in ml,, subtracted from the volume (ml.) of 0.1N HC1 required for titration of a control standardization of 25.0 ml. of 0.25N NaOH is the net volume of acid equivalent to the carbon dioxide. The calculation is carried out by the equation

N x net ml. of acid x 8.8 ^A'U'A' = wt. of sample (g.)

Stark's (51) method for the microdotermination of pectic substances in cotton based upon Dische's (12) carbazole reaction has been modified (34) as a result of a critical study cT the variables affecting the quantitative aspects of the reaction. The modified method shows promise for the determination of total pectic substances.

Deest6rify a 0.1 percent solution of pectin in 0.05N NaOH for 30 minutes at 25°C. and dilute to 0.002 percent. Add 2 ml. of this solution to 12 ml. of ice-cold concentrated sulfuric acid in a 25xl80-mm. test tube. Mix and heat the tube and contents in a boiling-water bath for 10 minutes. Cool the tube and contents to 20° C. and add 1 ml. of ethanol containing 1.5 mg. of carbazole. Mix thoroughly and after 25-5 minutes at room temperature, determine the color intensity with light of wave length 520 m^. A blank contain- ing the sample plus reagents (without carbazole) is used to set the colorimeter. A standard curve with 20 and 40 micrograms of galac- turonic acid hydrate (mol. wt. 212) is used to obtain the anhydro- uronic acid content (mol. wt. 176) of the pectic substance analyzed. Hexose sugars in two-fold excess, glucuronic acid, and mannuronic acid interfere.

Acetyl ; Sugar-beet pectin and perhaps others contain acetyl groups. Analysis for this functional group by simple alkaline saponification procedures followed by back titration does not yield satisfactory results. Clark's (9) method was therefore modified to be applicable to pectic substances (46). The procedure follows:

Weigh 0.5 gm, of pectin into a 250-ml. Erlenmeyer flask and add 25 ml. of 0.1N_ NaOH. Stopper the flask and stir the contents until the pectin is dissolved. Set the flask aside for at least 1 hour (overnight is also permissible), dilute the contents to 50.0 ml., and withdraw a 20.0-ml. aliquot. Place the aliquot in the distillation apparatus. Add 20 ml. of magnesium sulfate-sulfuric acid solution (100 gm. mag- nesium sulfate (crystal) and 1.5 gm. of sulfuric acid diluted to 180 mo.). Steam distill and collect about 100 ml. of distillate, keeping the volume in the distillation flask low. Titrate the acetic acid with 0.05N NaOH to a phenol red endpoint. A blank titration on water plus 20 ml. of the magnesium sulfate-sulfuric acid distilled as de- scribed amounts to less than 0.1 ml. of the standard alkali. The cal- culation is made as follows:

N x vol. of alkali (ml.) x 4.3

% acetyl = wt . of sample in aliquot (.gm. ) ( — OCCHj )

10

Viscosity; The contribution that pectin makes to jelly formation, to the viscosity of many food products, and to the firmness of texture is due in part to its molecu- lar weight. The simplest measure of viscosity average molecular weight is the intrinsic viscosity (Vj.). This is defined as the limiting value for the ratio

£ as C, the concentration, approaches zero, where r is the viscosity of the solution relative to the solvent. The intrinsic viscosity is indicated as f~ /? ~J , ^[ i* or' "^is re'Por^ » V±<* ~~

Pectin contains carboxyl groups, which, when ionized, contribute to the flow be- havior of pectin solutions, because electrostatic repulsion between them cause lengthening of the chain. This effect can be reduced by addition of either salt or acid, The procedure follows:

Weigh exactly 0.1 gm. of pectic substance (ash- and moisture-free basis). Dissolve in 50 ml. of water, adjusting the pH if necessary, Stir two hours. Add 0.80 gm. of sodium chloride and 0.200 gm. of Calgon or neutral Versene in 15 ml. of distilled water and stir another hour. Adjust the pH to 6.0+0.2. Rinse the electrodes into the solution and make up to 100.46 gm. or transfer to a 100-ml. volumetric flask and make to volume. Stir rapidly and thoroughly one minute and cover or stopper. If the solution is cloudy or contains dust or fibers, cen- trifuge in covered tubes or pass through a coarse sintered-glass filter. Determine the viscosity of the solution within an hour after the pH adjustment, by means of an Ostwald-Cannon-Fenske No. 50 pipet with 10 ml. of solution at 25+0.03° C. (A Hoeppler falling-ball type of viscometer can be used as an alternative method.) Determine the efflux time in the same instrument for the solvent, which is con- sidered to be 0.8 percent sodium chloride, 0.2 percent Calgon solu- tion. Because the density difference between solution and solvent is only 4 parts in 10,000, the relative viscosity is essentially equal to the ratio of the time of efflux for solution to that for solvent. Obtain the intrinsic viscosity directly from the curve in Fig. 1, which is based on Martin's exponential equation, in which the constant, k' , is assumed to be 0.40 (40) .

Maximum errors in intrinsic viscosity resulting from deviations in k' (l^) with citrus or apple pectic substances are not more than 0.06 at = 3.5 and 0.20 at

= 7.0. If greater accuracy is required, determine the relative viscosity at three concentrations, such as 0.15, 0.10, and 0.05 gm. per 100 ml., plot the ratio (?£r-l)/c against c on semi-log paper, and extrapolate to zero concentration to get the intrinsic viscosity.

Care should be exercizod in cleaning the viscometer. When in doubt, cleaning solution should be used. When a series of determinations is made on the same day, thorough rinsing five times with distilled water followed by a pure 95 percent ethanol or acetone rinse and suction drying is sufficient.

The degree of esterificat ion influences the value of the intrinsic viscosity. Our results agree with tho conclusion of Vollmert (53) that the intrinsic viscosity decreases with increasing esterification. The magnitude of this influence is still under investigation,, Inasmuch as most comparisons are made between pectins of similar ester content, viscosity can be a useful index of jelly grade or molecular

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weightv. Do not deesterify by alkali and measure the intrinsic viscosities of pectic acid in order to compare molecular weights of pectins (53, 54), The beta- elimination hypothesis indicates that the higher the degree of esterif ication the greater will be the degradation of pectin with alkali (54). Pure pect ine sterase might be used for this purpose, but the time required for complete reaction and subsequent purification is excessive.

Number Average Molecular Weight. The number average molecular weight is an im- portant factor controlling jellying with pectin. Many investigators recommend converting pectin to the nitrate, then measuring intrinsic viscosity in acetone solution, and relating that to molecular weight measured by osmotic pressure (16, 50). Direct measurement is preferable to reduce the chance of degradation and" of~~fract ionation. We use the Bull osmometer (5, 6) slightly modified as shown in Fig. 2. Instructions for its use follow:

Prepare solutions of pectic substances at three concentrations, pre- ferably below 0.60 percent (ash- and moisture-free basis), in 0.2M sodium chloride at pH 4.5. Solutions are preserved with 0.001 percent phenylmercuric nitrate. Introduce 0.5 ml. of toluene into the capil- lary of the osmometer. Fill the chamber of section A with salt solu- tion. Knot a 5-in. section of water-swollen uncoated cellulose sausage casing (wall thickness 20 microns, diameter, 12/32, 18/32, or 8/32 in.). Trim to proper length and tightly fasten to lower end of section B with a rubber band or linen thread. Test sac by immersion in water, and application of air pressure through top of section B. Remove excess water, rinse inside of sac with pectin solution, and fill to desired height, removing all air bubbles. The difference in levels in sections A and B should be near the estimated osmetic pres- sure of the pectin solution. It is helpful to mark these two levels on the tubes with wax pencil in advance. Grease joints with stopcock lubricant. Join sections A and B with stopcock open. Restore shape of sac and remove bubbles, if any, from region of stopcock by blowing on section B. Add salt solution to section A to the desired level. Remove any bubbles from toluene column by suction. Retard evaporation frcm tubes by covering them with small vials or slotted corks or stoppers. Immerse osmometer in water bath at 25C+0.01°C. After 30 minutes read liquid levels in all three arms by means of a catheto- meter or traveling telescope. Close stopcock. Read toluene and pectin solution levels every day until difference is constant. At least 5 days are required with commercially available tubing. (Before reading the toluene level it should be temporarily raised about 1 mm. by gentle suction. If the capillary is clean, the level will return to its undisturbed position in a few seconds.) Calculate the osmotic pressure from the equation:

P = 1.01 (Sx - Bi ) + 0.866 (Ti - T2) - ($i - S2)

where P is osmotic pressure in cm. of water at 4°C. ; is initial level of pectin solution; B-^ is initial level of salt solution; T-j_ is initial level of toluene; subscript 2 refers to final levels; 1.01 is the density of the pectin in sodium chloride solution as de- scribed above. Divide osmetic pressure by pectin concentration

2

o o

u

rx %

in

d

>-

< _j _i

< O

o

12 MM.O.D.

8MM.0.D.

STOP COCK 2 MM. BORE

22 MM.O.D.

24x 40 ▼

9.5MM.0.Da\ 4 MM. I.D.— »

2

o

-leg

to

o m

21 MM.O.D.

8 MM.O.D.' SECTION A

SECTION B

MODIFIED BULL OSMOMETER

FIGURE 2

-14-

expressed as percentage (gm. per 100 ml, of solution) end plot P/c vs. C. The intercept at C => 0 is the limiting value for the ratip p/c and is used in the calculation of molecular weight. If temperature during measurement is maintained at 25°C. , the molecular weight is calculated from the equation:

_ 25.3 x 104

Significant diffusion of pectin through the membrane or leaking past it can be detectedby testing the salt solution by means of carbazole (51 ) (page 9«

The osmometer must be clean so that capillarity differences will not introduce errors. If aqueous solution menisci do not have the same shape or if the toluene does not return to the same level after being raised repeatedly about 0.2 cm., clean the osmometer with "cleaning solution" before the next determination.

is advisable to measure duplicates on each concentration. If (S-^ - B-^ ) is set a little above the expected osmotic pressure in one determination and below it in another, a plot of apparent osmotic pressure vs. time will increase the accuracy and enable determination of the equilibrium value in a shorter period of time .

Jelly Grade: The most important direct application of pectin is as a gelling agent. To know how much pectin to add to a fruit juice to make a satisfactory jelly, the jelly grade must be known. This is defined as the number of grams (or lbs.) of sugar with which one gram (or lb.) of pectin will form a 65 percent soluble-solids gel of specified strength under suitable acid conditions* No standard amount of acid nor strength value has been accepted; consequently there is likely to be variation in jelly grades of pectins purchased from different companies. Various methods for jelly-grade determination have been developed. The English use the BAR tester, devised by Campbell (7). The Germans use the Luers-Lochmuller tester (31). In this country the Tarr-Baker tester (52) and the ridgelimeter (ll) have met with favorable response. Methods of grading pectin are under examination by a committee of the Institute of Food Technologists with the objective of establishing a standard method. Methods of preparing jellies vary considerably and only a modification of the simplest (ll) will be described

Weigh 5.20 gm. of pectin (assume 150-grade as a trial), mix with 50 gm. of sugar and dissolve in 400 ml. of water. If the pectin does not dissolve, add a small amount of Calgon. Adjust the pH to 3.2 + 0,1. Pour into a tared jelly pan (2 qt . ) , adjust net weight to 52F gm. , adding water rinsings of the beaker Yfhich contained the pectin. Bring to a boil in less than 5 minutes, with stirring. Start timer and slowly add 725 gm. of sugar with stirring. Stir and boil for 2 minutes after addition of last portion of sugar to insure complete solution of pectin. Total time of heating at or near boiling should not exceed 5 minutes. At 4 minutes and 30 seconds the weight should bo checked and the net weight brought to 1200 gm. either by evapor- ating more water or adding some. Bring to boiling again and at the end of 5 minutes pour the solution into jelly glasses. The glasses hr.ve a known depth (7.95 cm. high inside 11, 23) and should contain

-15

2 ml, of acid solution (l gm. citric acid mcnohydrate to 1 ml. of water) whon the pectin solution is poured. The glasses should have sideboards 3/4 in. above edge of glass. Scotch or drafting tape can be used for this purpose.

Stir contents of the glasses vigorously for 1 or 2 seconds and set aside for 24 hours. Slice off the jelly flush with top of glass. Remove jelly from glass and place it bottom side up on a clean, dry platform of the ridgelimeter . Measure the percentage of sag after 2 minutes and read the deviation from assumed, grade (23). If the assumed grade was off by more than 20 percent, make another jelly closer to specified strength. The pH of the jelly should be 2 e U P r* less .

The torsion tester yields the same results (42) as does the BAR tester (38) and offers the advantage of yielding fundamental values if a cylinder is used instead of a blade. The ridgelimeter can be used, to obtain modulus of elasticity in com- pression if strong jellies are cast in the form of cylinders.

Jelly grade can be determined from the intrinsic viscosity, by means of the curve shown in Fig,, 3. Further work will be necessary to establish its reliability, because it represents data from only a few citrus pectins, and molecular weight heterogeneity probably has a marked influence on the curve.

Points that should be mentioned: The pectin must be completely dissolved before heating. Any pectin precipitated by the added sugar should be redissolved before the gelling solution is poured into glasses. The temperature of storage should be near 23° to 25°C, especially with slow-set pectins (28). Rate of application of force should be constant. Only small displacements should be applied for calcu- lations of elastic moduli. One advantage of the sag tester and rigidometer is that the force is applied, for a constant period of time for each jelly. Displace- ments measured under these conditions are more likely to yield fundamental con- stants than a breaking test in which force must be applied until breaking results. The latter, of course, causes more yielding of the gel structure. One result is that grades measured by the two types of testers seldom agree.

It has been stated (8) that it is unsound to measure fundamental constants of pectin jellies. Such measurements have been made even on solutions of polymers by applying force over very short periods of time (l). Similarly if displacements of pectin gels are small, they are also essentially linear with respect to applied force (37, 41), and calculations of elastic moduli are reliable. The arguments and data offered by Cheftel and Mocquard (8) indicate that breaking strength measurements are not reliable; on the other hand Bender (2), by carefully control- ling each step, reports that the Tarr-Baker tester gives reproducible results.

No mention has been made of grading low-methoxyl pectins. We generally measure methoxyl content and intrinsic viscosities to establish a value for comparison of such pectinic acids. Generally a pectin prepared by the method used in this Lab- oratory (41), with a uniform methoxyl content of 3.2 + 0,3 percent and an intrinsic viscosity of 3.4 + 0.5, will be satisfactory for gel purposes. For films and fibers the lower limit for viscosity is about 3.5. Higher values of methoxyl con- tent require additional calcium ion for gelation, while higher values of viscosity require less calcium. By varying calcium ion concentration, pectinic acids out- side the mentioned range can be applied for formation of gels, etc.

JELLY GRADE BY R IDGELI METER

— — ro rv> 01

ui o 01 o ui o

o o o o o o o

-17-

Setting Time; The setting tirre of pectins is of importance to jam, marmalade, and preserve processors. Several methods have teen proposed to measure this factor (18, 23, 39). Rate of cooling, presence of rtetal ions, pH, solids content, etc. influence setting time and temperature (18, 41) . We have selected, tut not tested, a simple method (23), which involves no change in technique from that used for preparing jellies for grading.

Prepare jelly as before and start a timer when filling a sample glass. Place the glass in a bath maintained at 30°C. so that the glass is surrounded almost to the top. At intervals give it a slow twist. When tho jelly at the top just congeals, stop the timer to obtain the set- ting time. Gelation can bo observed by slowly turning the glass. "When tho turning motion ceases, a liquid system will continue to rotate in tho same direction. A gel system will show elasticity and move in the opposite direction. Rapid-setting pectins will gel in less than 2 minutes, while slow-sotting pectins require mere than 3 minutes.

Usually a high-molocular-weight pectin with 10.5 percent or more ofmethexyl groups (degree of esterif ication (DE) above 70 percent) will be a rapid-set pectin; those with 7 to 10 percent mothoxyl (DE 50 to 70 percent) will bo slow-set pectins. At lower DE's the pectin will tend to be rapid-set; ?lso the presence of polyvalent cations drastically decreases the setting time.

Optical Rotation; Tho rotation of polarized light by pectin solutions is a characteristic property which can be usod to determine pectin when the specific rotation is known (36 ) . This value is measured by dissolving 0.5 gm. of pectin in 75 ml. of water, adjusting the pH to 4.5 to 7.0, and making to 100 ml. or gm, Tho solution is poured into a 2-dm. tube, and the optical rotation is measured in a polarimeter or saccharimoter at 25°C. ^ith a saccharimeter which has a Bates- Jackson scale, the specific rotation is obtained by the equation,

>- obs. rot. x 100 x 0.346

M ' —c

Pectin from oranges has a specific rotation of +230 when the mothoxyl content is about 10 percent. To apply this to the determination of pectin in extracts of orange peel, tho following procedure is used.

Filter 100 ml. of the solution containing about 0.5 percent pectin with filter aid, discarding the first 25 ml. Measure the optical rotation of the filtrate in a 1-dm. tube. To 25 ml. of this solu- tion add 25 ml. of copper sulfate solution. (The copper sulfate solution contains 9.4 gm. of the pentahydrate, 27.2 gm. of sodium acetate trihydrate, 12 ml. of glacial acetic acid made to one liter.) Filter the precipitated copper pectinate and measure the rotation of the filtrate in a 2-dm. tube. Tho difference between this and the rotation of the pectin extract is the net rotation due to the pectin alone and is used in tho calculation.

% pectin

net rotation x 0.346 x 100

230

Yields by this method are higher than those by uronide determination.

-18-

5/

Pectic Enzymes-/

Changes in the nature of pectin will occur in unblanched purees, macerated, or broken tissue through action of pectic enzymes present. These changes occur even in the frozen state. It should be mentioned that the enzymes increase in activity with increase in temperature; therefore during inactivation by heat there is a period when pectic enzymes are very active. Pectinesterase deesterifies pectin, making it susceptible to precipitation by calcium cr other polyvalent cation. Juice clouds settle and frozen orange concentrates gel. Pectinases dogrado pectin to galacturonic acid or low-molecular-weight polymers of it. Viscosity changes occur and in this caso, too, juice clouds will settle. Measurement of the activ- ities of these two typos of onzymes provides valuable clues to the solution of many problems involving gelling, viscosity changes, synerosis, and cloud instability. The methods for such measurements follow:

Pectinesterase (3£)) (Pectase, Pectin Methylosterase ) ; Pectinesterase in most plant tissues is rarely in solution but is adsorbed on the insoluble cellular solids. It is then necessary to extract this material in 0.25M NaCl and maintain a pH of 8 for about 1 hour while the enzyme dissolves. The assay for its activity is carried out as described (52 ) .

Add 2 ml. of 1„5M NaCl to 10 ml. of 1 percent pectin solution, stir with carbon dioxide-free air and titrate to pH 7.5 with 0.02N NaOH. Use a constant- temperature bath to maintain a temperature of 30°C. and add enzyme and water to adjust the volume to 20 ml. Immediately record the time and volume of alkali required to maintain the pH at the constant value. Adjust the concentration of enzyme to require about 1 to 3 ml. of 0.02N alkali in 10 minutes. The results can be expressed in pectinesterase units (PE.Uc/gm. , the expression for milliequivalents of ester hydrolyzed per minute per gram of enzyme).

Polygalacturonase (PG): Polygalacturonase (sometimes considered synonymous with pectinase ) seldom occurs in higher plants, and then only in small amounts. It is produced by many bacteria and fungi and has been reported in snails. Little in- formation exists on extraction of the enzyme from plants, but its assay can be conducted on plant extracts or macerates as described. Although a quantitative method, based on viscosity changes, would be more desirable, our experience has been based on changes of reducing value as presented below (21).

Add 1 ml, of enzyme solution of the proper dilution to 99 ml. of 0.5 percent solution of pectic acid at pH 4 and at 25°C. Remove aliquots of 5 ml. and add to 0.9 ml. of 1M sodium carbonate in a glass-stoppered flask. Add 5 ml. of 0.1N iodine, stir thoroughly, and after 20 minutes acidify with 2 ml. of 2M sulfuric acid and titrate the residual iodine with 0.05N sodium thiosulfate solution. A calibration curve prepared with galacturonic acid monohydrate is used to obtain the activity. Under these conditions, 1 milliequivalent of reduced iodine corresponds to 0.513 millimole of aldose liberated. The activity is conveniently ex- pressed as (PG.u.)/ml., or the millimoles of reducing groups liberated per minute per milliliter of enzyme.

5/ Nomenclature is listed on page 2h»

-19

Other Poetic Snzymes: Other pectic enzymes are mentioned in the literature (28, 30, 45, 48), but for the most part they are not so well characterized as PE and PG; consequently no method of assay is recommended for them. The depolymeraso activity discovered by McCclloch and Kertesz (33) can be measured in the same way as PG activity with pH adjusted to 4.5. The polymethylgalact uronase activity de- scribed by ^eogmiller and Jansen (48 ) we s also measured in the same way as PG, except that citrus pectin at pH 6.0 was used.

Literature Cited

(1) Ashworth, J. N., and Ferry, J. D.

1949. Mechanical Properties of Substances of High Molecular Weight. V„ Rigidities of Polyisobutylene Solutions in Various Solvents. Jour. Aner. Chem. Soc. 71:622-628.

(2) Bender, W. A.

1949. Grading Pectin in Sugar Jollies. Analyt. Chem. 21:408-411.

(3) Berglund, D. T,

1950. Methods for Characterization of Pectins. Sooker 6 (13) : 219-223.

(4) Eonner, J.

1950. The Pectic Substances. Plant Biochemistry. Academic Press, Inc., New York, p. • 99.

(5) Bull, H. B.

1941. Osmotic Pressure of Egg Albumin Solutions, Jour. Biol. Chem. 137: 143-151.

(6) Bull, H. B., and Currie, B. T.

1946. Osmotic Pressure of Beta-Lactoglobulin Solutions. Jour. Amer. Chem. yoc. 68:742-745.

(7) Campbell, L. E.

1938. The Calibration of Jelly-Testers. Jour. aoc. Chem. Ind. 57:413-417.

(8) Cheftel, H. , and Mocquard, J.

1947. Contribution to the Study of the Rheological Properties of Pectin Jellies. Jour. ^oc. Chem. Ind. 66:297-298.

(9) Clark, E. P.

1943. Semimicrc Quantitative Organic Analysis. Academic Press, Inc., New York, p. 73.

(10) Committoe on Nomenclature of Pectin of the Agriculture-Food Division,Amsrican

Chemical ^ociety.

1927. Jour. Amer. Chem. S0c. (Proc.) 49:37.

(11) Cox, R. E., and Higby, P. H.

1944. A Better lay to Determine the Jelling Power of Pectins. Food Indus. 16:441-442.

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Dische, Z.

1947. A New Specific Color Reaction of Huxuronic Acids. Jour. Biol. Chem. 167:189-198.

Bggenberger, T)?.

1949, Bicchemische Untersuchungen an Apfeln wahrend der Entwicklung und Lagerung. Ber. schwoiz. botan. Ges. 59:91-154.

Ehrlich, (jF.

1932. IJber die( Chemie des Pektins. II. Eine typische Reaktion der d-Galakturon e aure und des Pektins. Ber. 65B:352-358.

Henglein^ F. A,

1943. IJber Protopektin und Protocellulose . J. Makromol. Chem. Series 3. 1:121-130.

Henglein, F. a,

1947. Nomenclature, Stoichiometry and Analysis in Pectin Chemistry. Makromol. Chem. 1:70-84.

Hinton, C. L.

1940. Fruit Pectins. Chemical Publishing Co., New York, p. 27. Hinton, C. L.

1950. The Setting Temperature of Pectin Jellies. Jour. Sci. Food & A-gr. 1:300-307.

Hirst, E. L., and Jones, J. K. N.

1946. The Chemistry of Pectic Materials. Advances in Carbohydrate Chemistry, Vol. 2. Academic Press, New York, pp. 235-251.

Jansen, E. f., Waisbrot, s. W., and Reitz, E.

1944. Errors in the Zeisel Methoxyl Values for Pectin Due to Retained Alcohol. Indus, and Engin. Chem., Analyt. Ed. 16:523-524.

Jansen, ii. F., and MacDonnell, L. R.

1945. Influence of Methoxyl Content of -^ectic Substances on the Action of Polygalacturonase. Arch. Biochem. 8:97-112.

Johnson, C. M.

1945. Determination of Water in Dry Food Materials. Indus, and Engin. Chorn., Analyt. Ed. 17:312-316.

Joseph, G. H. , and Baier, W. E. 1949. Methods of Determining the Firmness and Setting Time of Pectin Test Jellies. Food Technol. 3:18-22.

Joslyn, 11. A., end Phaff, H. J.

1947. Recent Advances in the Chemistry of Pectic Substances. Yfallerstein Lab. Commun. 10:39-56.

-21-

Kertesz, Z. I., Baker, C. L. , Joseph, G, H, , Mottern, H. H. , and Olsen, A. G

1944. Report of the Committee for tho Revision of the Nomenclature of Pectic Substances. Chem. and Engin. News 22:105-106.

Kertesz, Z. I.

1951. The Pectic Substances, Interscience Publishers, New York. 628 pages

Kertesz, Z. I., and McColloch, R. J.

1950. The Pectic Substances of Mature John Baer Tomatoes. New York Agr. Expt. Sta. Bull. No. 745.

Kertesz, Z. I., and McColloch, R. J.

1950. Enzymes Acting on Pectic Substances. Advances in Carbohydrate Chemistry, Vol. 5, Academic Press, Inc., New York, pp. 79-102.

Lefevre, K. TJ., and Tollens, B.

1907. Untersuchungen uber die Glucuronsaure , ihre Quantitative Bestimmung und ihre Farbenreaktionen. Ber. 40:4513-4523.

Lineweaver, H., and Jansen, E. F.

1951. Pectic Enzymes. Advances in Enzymology, Vol. 11. Interscience Publishers, New York, pp. 267-295.

Luers, H., and Lochmuller, K.

1927. The Measurement of the Jelly Strength of Fruit Pectins. Kolloid Zeit. 42:154.

MacDonnell, L. R., Jansen, F. , and Lineweaver, Hans.

1945. The Properties of Orange Pectinesterase . Arch. Biochem. 6:389-401.

McColloch, R. J., and Kertesz, Z. I.

1948. An Unusually Heat-Resistant Pecto lytic Factor from Tomatoes. Arch. Biochem. 17:192-199.

McCcmb, Elizabeth A., and McCready, R. M.

1952. Colorimetric Determination of Pectic Substances. Submitted to Analyt. Chem.

McCready, R. M. , Swenson, H. A., and Maclay, W. D.

1946. Determination of Uronic Acids. Indus, id Engin. Chem., Analyt. Ed. 18:290-291.

McCready, R. M. , Shepherd, A. D., Swenson, H. A., Erlandsen, Roberta F. , and Maclay, D.

1951. Determination of Citrus Pectic Substpnces by Optical Rotation. Analyt. Chem. 23:975-977.

Neukom. J.

it

1949. Studion uber das Geliemvermogen von Pektinstof f en und anderen hochmol3kularenitPolyoxy v arbindungen , Dissertation, Teohnisohen Hochschulen. Zurich, Switzerland.

22-

Olliver M. • '■

1950. Laboratory Assessment of Pectin Quality with Special Reference to Jelly Grading, Jour. Sci. Food & Agr, 1:329-336.

Olsen, A, J,s Stuewer, ^. F. , Fehlborg, E. R., and Beach, N, M. 1939. Pectin Studies. Indus, and Engin. Chem. 31:1015-1020.

Owens , H. S., Lotzkar, H., Schultz, T , H., and Maclay, W. D.

1946. Shape and Size of ^ectinic Acid Molecules Deduced from Viscometric Measurements. Jour. Amer. Chem. Soc. 68:1628-1632.

Owens, H. S., McCready, R. M. , and Maclay, W, D.

1949. Gelation Characteristics of Acid-Precipitated Pectinates. Food Technol. 3:77-82.

Owens, H. S., Porter, 0., and Maclay, If. D.

1947. New Device for Grading Pectins. Food Indus. 19:606-608.

Pallmann^H.

1944. Schweiz. Landw. Monatshefte 22:306, 334 (cited from Ref s .'.13 and 44),

Pallmann, H., and Deuel, H.

1947. Ubersicht uber die Chemie und Physik der -^ektinstoffe und Besprechung der neueren Literatur 1937-1946. Chimia 1:27-33, 51-56.

Phaff, H. J., and Joslyn, M, A.

1947. The Newer Knowledge of the Poetic Enzymes. Waller stein Lab. Commun. 10:133-148.

Pippen, E. L. , McCready, R. M, , and Owens, H. S.

1950. Determination of Acetyl in Pectin. Analyt. Chem. 22:1457-1458.

Preston, R. D.

1949. The Organization of the Cell Walls in Plants in Relation to the Structure of Plants, Fibre Science. The Textile Institute, Manchester, England, pp. 218-246.

Soegmiller, C, q, f and Jansen, E. F.

1952. Polymethylgalacturonase , an Enzyme Causing the Glycosidic Hydrolysis of Esterifiod Pectic Substances. Jour. Biol, Chem. 195:327-336.

Shriner, R. L.

1948. Quantitative Analysis of Organic Compounds, 3rd Edition, Edwards Brothers, Inc., Ann Arbor, Mich., p, 35.

Speiser, and Eddy, C . R.

1946. Effect of Molecular Weight and Method of Deesterif ication on the Gelling Behavior of Pectin. Jour. Amer. Chem. Soc. 68:287-293.

Stark, S. LI, , Jr.

1950. Determinat ion of Poetic ;S-ib stances in Cotton. Analyt. Cham. 22 1158-1160.

Tarr, L. W.

1926. Fruit Jollies, Jelly Strength Measurements. Delaware Agr. Expt Sta. Bull. No. 142, 33 pp.

Vollmert, B.

1950. Viscositet unci Verostorungsgrad bei Pekt inlosungen. Lfekromol. Chem. 5:128-138.

Vollmert,, B.

1950. Uber den alkalischen Poctinabbau, Makromol. Chem. 5:110-127*

-2h-

Nomenclature

The American Chemical Society has recommended the following nomenclature (25):

Pectic Substances, "Pectic substances" is a term applied as group designation for those complex colloidal carbohydrate derivatives which occur in or are prepared from plants and contain a large proportion of anhydro-galacturonic acid units -whicl are thought to exist in a chainlike combination. The carboxyl groups of poly- galacturonic acids can bo partly estorified by methyl groups and partly or com- pletely neutralized by one or more bases.

Protopect in. The term "protopectin" is applied to the water-insoluble parent pectic substances which occur in plants and which upon restricted hydrolysis yield pectin or pectinic acids.

Pectinic Acids. The term "pectinic acids" is used for colloidal polygalacturonic acids containing more than a negligible proportion of methyl ester groups. Pectinic 1 acids under suitable conditions are capable of forming gels with sugar and acid or, if suitably low in methoxyl content, with certain metallic ions. The salts of pectinic acids are either normal or acid pectinates.

Pectin. The general term "pectin" (or pectins) designates those water-soluble pectinic acids of varying methyl ester content and degree of neutralization which are capable of forming gels with sugar and acid under suitable conditions.

Pectic Acids . The term "pectic acids" is applied to pectic substances mostly com- posed of colloidal polygalacturonic acids and essentially free from methyl ester groups. The salts of pectic acids are either normal or acid pectates.

The Committee on Nomenclature is acutely awar3 of the difficulties connected with the task. They stressed the fact that poetic materials represent mixtures of polygalacturonides with variable molecular weights and, with the exception of pectic acid, may also contain different proportions of methyl ester groups in the individual polygalacturonic acids. Acid salts may contain polymer units with differ- ent proportions of metallic ions, and somotimos several species of cations. Those complexities must be borne in mind when solutions to pectin problems are sought.

Pectic Enzymes . The nomenclature for pectic enzymes has not been changed since 1926 (10 ) , but a recently proposed nomenclature defines them as follows- (50) :

Protopect inase is a name used for the enzyme that converts protopectin into a soluble product; other names are pectos inase and propect inase . The existence of protopectin or protopect inase is not firmly established.

Pectinesterase (PE) is the enzyme that catalyzes the hydrolysis of the ester bonds of pectic substances to methanol and pectic or pectinic acids; other names used are pectase, pectin demethoxylase , pectin mothoxylase, pectolipaso, and pectin met hylest erase .

Polygalacturonase (PG) is the enzyme that catalyzes the hydrolysis of glycosidic bonds between doesterified galacturonide residues in pectic substances; other names aro poet inase and pectin glycosidase.