Measurement of the Starch Content of Commercial Starches

Analytical Working Party of the Starch Experts Group (STEX) of the European Starch Associations (ESAY

For the EC starch regulation 2169/1986 a minimum purity of 97% starch on dry substance is required to obtain the new end-use refunds. Three different methods for the determination of starch were tested. Two polarimetric methods (EC L123/72, AOAC 14031-032) and an enzymatic merhod (EC L158/86) were used to determine the high starch content of commercial unmodified star- ches. The results of a ring test between six laboratories shows that the AOAC method, solubilizing the starch in the presence of calcium chloride, has the smallest standard deviation. An explanation for the differences observed between the three methods is given, based on the sugar spectrum of the hydrolyzed starch. An independent ring test in France shows a modified enzymatic method, developed by AFNOR (the French standards organization) produces satisfactory results.

Messung des Starkegehaltes handelsublicher Starken. In der EG-Starkeregelung 2169/1986 wird eine Mindestreinheit von 97% Starke in der Trockensubstanz gefordert, um die neue Endver- brauchs-Erstattung zu erhaltcn. Fur die Bestimmung der Starke wur- den drei verschiedene Methoden getestet. Zwei polarimetrische (EC L123/72, AOAC 14031 -032) und eine enzymatische Methode (EC L158/86) wurden angewendet, urn den hohen Starkegehalt handels- iiblicher nichtmodifizierter Starken zu ermitteln. Die Ergebnisse einer Ringuntersuchung zwischen sechs Laboratorien zeigen, daR die AOAC-Methode, bei der die Starke in Gegenwart von Calciumchlo- rid gelost wird, die geringste Standardabweichung hat. Auf der Grundlage des Zuckerspektrums der hydrolysierten Starke. Ein davon unabhangiger Ringtest in Frankreich zeigt, daR eine von AFNOR (die franzosische Standardisierungs-Organisation) modifi- zierte enzymatische Methodc zu befriedigenden Ergebnissen fuhrt.

1 Introduction On 1st July 1986 a new E C starch regime came into force whereby substantial refunds are given for starch used in certain agncultur- ally unprotected applications (EC regulation 2169/86). In order to qualify for the new end-use refunds, a minimum purity of 97% starch o n a dry substance basis is required. Consequently it is essential to have an analytical method that allows the measurement of this high starch content in a reliable, reproducible and accurate way. The Starch Expert Committee of the European Starch Associations decided to take a critical look at existing methods. All the methods chosen were inten- ded to some extent to measure the relatively low starch content of various starch containing products. To meet the high purity requirements of 97% on a dry basis, the standard deviation of the applied method should be less than 1%. To evaluate the accuracy and repeatability between different laboratories, the Starch Industries organised a ring test amongst some of its members. The methods tested were: 1) Ewers method EC L123172 2) Calcium chloride method 3) Enzymatic method E C L1.58186 The methods were evaluated by analysis of the starch content of wheat starch, potato starch and maize starch (Tables 1 and 2). The analyses were carried out in triplicate by 2 or 3 analysts within one laboratory and repeated on the same samples by different laboraties. The results were collected and the statistics were calculated according to the A O A C Statistical Manual [ 11.

A O A C 14031-32

2 Principles

2.1 Ewers method EC L123/72 The method consists of a double determination. The sample is first heated with diluted hydrochloric acid. By heating, the starch is gelatini- zed and by the action of hydrochloric acid the starch is hydrolyzed to smaller oligosaccharides and glucose as is shown in Fig. 1. After clarification and filtration the rotatory power is measured by polarime- try. Secondly, a sample is extracted with 40% ethanol. The

Table 1. Starch Content (dry basis) of Commercial Starches (%).

Method Wheat Maize Potato

Ewers 0 0 OX

CaCI? 0 0 ax

Enzymatic 0 0 OX

98.94 0.36 2.07

97.26 0.30 1.11

97.45 1.53 4.06

98.67 0.48 1.41

97.84 0.28 0.83

97.68 1.84 3.69

100.68 0.58 1.73

98.88 0.40 1.05

99.78 I .46 4.90

00 = repeatability standard deviation O X = reproducibility standard deviation

Table 2. Non-Starch Components Perccntages on Dry Basis.

Product Method Protein Ash Total lipids

IS0 3188 I S 0 3593 after hydrolysis CIRF 8.14

Wheat starch 0.16 0.25 0.31 Maize starch 0.33 0.07 0.51 Potato starch 0.10 0.21 0.02

filtrate is acidified with hydrochloric acid and after clarification and filtration, the rotatory power of the free sugar content is measured under the same conditions as in the first determination. The starch content is given by difference between the two measure- ments, multiplied with a known factor. This factor depends on the nature of the starch: For corn starch the factor F = 184.6, for wheat starch F = 182.7 and for potato starch F = 195.4.

* Participants: Amylum, Avcbe, Cargill, CPCE. Roquette, ZBB.

41 4 starch/starke 39 (1987) Nr. 12. S. 414-416 0 VCH Verlagsgesellschaft mbH. D-6940 We~nheim. 1987 0038-9056/87/1212-0414$02.50/0

3.2 Calcium chloride AOAC 14031-032

Flg. 1 Fig 2 Fig. 3

Figure 1-3. Sugar analyses were performed on a Bio-Rad HPX-87C column at 85°C with water as eluent and a flow rate of 0.4 mYmin. I . Glucose 2. Maltose 3. Triose 4. Tetraose 5 . Pentaose 6. High molccular weight polysacharides 7. CaCI2 8. Acetic acid 9. Sodium salts.

2.2 Calcium chloride method (AOAC 14031-32)

The calcium chloride method is a polarimetric method for the determi- nation of the starch content. The method is based on a differential measurement of the optical rotation of the ,,treated" sample and on an ethanol extracted blank. The calcium chloride method, unlike the Ewers method is based on the solubilisation of the starch in a 33% calcium chloride solution at neutral or slightly acidic pH by the addition of acetic acid. The high ionic strength causes the starch molecule to unfold and to dissolve without the formation of oligosaccharides or glucose (Fig. 2). The free sugar content, present in the sample before treatment. can be detected by an ethanol extraction. In the ringtest this has been deleted.

2.3 Enzymatic method EC L158/86

This method is based on the hydrolysis of the starch by an amyloglucosi- dase, at 60°C, of which the endproduct is glucose (Fig. 3). Before hydrolysis the starch is dispersed in a 0.5 N sodium hydroxyde solution at 60°C. The glucose content, after hydrolysis, is determined by an enzyme oxidation-reduction system, detected at U. V. wavelengths.

3 Critique of the Methods

3.1 Ewers EC L123/72

This method relies on hydrolysis of starch by hydrochloric acid at atmospheric pressure using a boiling water bath for 15 min. Under these conditions hydrolysis of the starch is quite incom- plete [2,3]. Therefore the degree of hydrolysis will depend on: - Heat transfer characteristics of water batidflask system used.

For instance the thickness of the flask, the extent of agitation of the contents of the flask, the thermal capacity of the water bath.

- The precision of timing of the hydrolysis before cooling. - Heat transfer characteristics of the flask during cooling [4,5,

- Influence of clarification reagents and their amounts [7, 81.

Thus, the preparation of the sample for polarimetry presents a number of sources of error. The polarimetric measurement itself is reproducible between laboratories, and standardization using sucrose is recommended.

61.

As the solution of the starch does not rely on hydrolysis but rather on the formation of a soluble complex, the heating step is much less critical. The preparation of the sample for polarime- try is straight forward and the polarimetric measurement is reproducible between laboratories. The factor used in the calculations is perhaps inappropriate for high starch concentrations. This explains the systematic low results.

3.3 Enzymatic EC L158/86

The benefit of an enzymatic method is that enzymes are specific in their action and, therefore, in the case of starch, hydrolyse only the starch and not other material present. In the present case we have nearly 100% starch so the specificity is less important. Indeed it is a disadvantage because amyloglucosi- dase, for instance, is not suited to break all 1,6 linkages. The alcohol content must be as low as possible before enzyme is added or the amyloglucosidase will be denatured. The reagents used are biologically unstable and this can cause problems in the hands of inexperienced operators. The use of fresh reagents is highly desirable but not always possible. At the end of the procedure 0.1 ml of the test solution is taken and this very small aliquot is a further source of error. Also the standardization of spectrophotometers between laboratories is notoriously difficult. Dissolving starch in a 0.5 N sodium hydroxide solution at 60°C is very difficult and could cause errors in the end determination.

An improved enzymatic method was tested by the French starch producers. This method combines parts of the official E C method with a French AFNOR method. The new method is proposed as a new AFNOR project V.036.06. Reproducible results were obtained this way (Table 3).

Table 3. Enzymatic Determination of YO Starch Content (dry basis) According to the AFNOR Method (Project V.036.06).

Method Wheat Maize Potato

AFNOR 97.78 98.60 98.36 ( 3 X 0.47 0.34 0.91

4 Conclusion

An examination in detail of the results of the collaborative test (which will be reported in detail in due course) indicates that of three methods studied, the calcium chloride method has the best reproducibility. It is also a simple method to carry out and needs a minimum of equipment. It must be remembered that none of the methods tested has been devised for the determination of starch contents at such high levels. The E C enzymatic and Ewers methods were originated for measuring the starch content of animal feeding stuffs and similar products with 20 to 50% starch content on dry basis. The calcium chloride method is intended for the determi- nation of the starch content of flour, that is up to a maximum of 85% starch on dry basis. It is a principle of regulatory bodies that a subtance refered to in legislation should be the compo- nent to be determined, whenever possible. The results obtained by the calcium chloride method are low in relation to the known starch content of the samples tested. It is likely that this is due to the factors used in the calculation of the results. The coefficients

starchktarke 39 (1987) Nr. 12, S . 414-416 415

of variation of the Ewers and enzymatic methods as currently written are unacceptably high. Due to the economic signifi- cance of starch purity determinations, a method is required which will give clear. indisputable results. In relation to a purity requirement of 97%, no method with a repeatibility between 2% and k 5% is likely to meet this requirement. The results obtained with the proposed AFNOR enzymatic method show that improvements can and should be made before a final analytical method can be agreed upon.

Bibliography

[ l ) Youden, W. J.,and H . Sreiner: AOAC Statistical Manual 1975. [2] Ewers, E. : Ztschr. f . offentliche Chemie 14 (1908), 150-157. [3] Witikler, S., and G. Luckow: StarchlStarke 19 (1967), 110-115. [4] Dudas, F.. Starch/Starke 28 (1976), 127-129.

F . : StarcNStarke 26 157-159. [6] Dudas, F.: StarchEtarke 23 (1971), 390-393. [7] Dudas, F.: StarchlStarke 24 (1972), 367-369. [8] Dudas, F.: StarchlStarke 22 (1970). 385-388.

Correspondence to: MI. E. Moreels. Amylum N . V., Burchtstraat 10, B-9300 Aalst (Belgium).

(Received: May 6, 1987).

Vergleichende Untersuchungen zur Erbsen- starkeisolierung auf nantechnischem Wege*

N. U. Haase, W. Kempf, G. Tegge und U. D’heur, Detmold

Kornererbsen enthalten Starken, die abhangig von der Sorte iibcr sehr hohe Amyloseanteile verfiigen. Fur die bisher weitgehend feh- lenden analytischen Untersuchungen derartiger Starken miissen vorab moglichst gute Isolierungen durchgefuhrt werden. Deshalb wurden in der vorliegenden Untersuchung die drei Verfahren der Flutung, der Zentrifugation und der Extraktion einander gegeniiber- gestellt, wobei die Proteinabtrennung besonderes Gewicht einnahm. Ein kurzzeitiger Kontakt der vermahlenen Erbsen mit Natronlauge fuhrte sowohl bei der Flutung als auch bei der Zentrifugation zu einer vergleichbar hohen Starkequalitat, doch lag der Wirkungsgrad der Starkeabtrennung bei der Flutung um 30% hoher. Die Extrak- tion fie1 gegeniiber den beiden anderen Methoden beziiglich der verwendeten MeRkriterien deutlich ab. Ein zusatzlich vorgenomme- ner Sortenvergleich wies darauf hin, daR die Starke aus Palerbsen besser abtrennbar ist als die aus Markerbsen.

Comparative Investigations on the Wet-Separation of Pea Starch. Peas contain starches with partly hrgh amylose fractions. As exact analytical investigations do not exist yet, the first thing is to develop an efficient technique to isolate these starches. Therefore, in this study three laboratory procedures of wet milling which are based on settling tables, centrifugation and extraction are compared. In this connection the efficiency of protein separation was of importance. A short contact of the ground peas with sodium hydroxide resulted in a comparably high starch quality when using tables as well as cen- trifuges. However, the recovery rate of starch was 30% higher when using tables. Compared to the other procedures the extraction method led to starches of much lower quality with regard to the tested quality criteria. An additional comparison of different pea species indicated that the starch separation from smooth peas is more efficient than that from wrinkled peas.

1 Einleitung Die sich verscharfende Situation der Agraruberschusse der Europaischen Gemeinschaft hat bereits zu einer Ausweitung des Anbauspektrums der Kulturarten gefiihrt. Mit der Bereit- stellung neuartiger Rohstoffe fur die industrielle Nutzung sol1 dabei eine Entspannung der Uberschurjsituation auf dem Nah- rungs- und Futtermittelsektor erreicht werden. Diese Sachlage tnfft neben verschiedenen 0 1 - und Faserpflanzen auch einige Leguminosenarten, bei denen es Ansatze gibt, neben dem Proteinanteil auch die Starke zu nutzen [l, 2 , 31. In der industriellen Verwertung finden iiberwiegend Starken Verwendung, die Amylosegehalte zwischen 20 und 25% auf- weisen. Der Einsatz amylosereicher Starken, die heute noch ausschlierjlich aus Amylomais s taqmen [4] bzw. uber Fraktio- nierungsschritte angereichert werden [ S ] , findet hingegen trotz vielfaltiger Einsatzmoglichkeiten [6, 7, 81 nur in sehr begrenz- tem Umfange statt. Die verstarkte Nutzung dieses Potentials wird voraussichtlich auch allein dann erfolgen, wenn es gelingt,

* Nr. 5579 der Veroffentlichungen der Bundesforschungsanstalt fiir Getreide- und Kartoffelverarbeitung. Detmold.

** Vortrag von N . U. Haase anlaBlich der 38. Starke-Tagung der Arbeitsgemeinschaft Getreideforschung in Detmold vom 22. bis 24. April 1987.

derartige Starken ohne vorgeschaltete Aufarbeitungstechniken zu nutzen. Als Rohstoff konnen dabei neben Amylomais [4] verschiedene Leguminosenarten [9] eingesetzt werden, die - sortenabhangig - ebenfalls sehr hohe Amylosegehalte be- sitzen. Eine Eingliederung dieser Leguminosenstarken in das vorhan- dene Starkeangebot erfordert in jedem Fall Starken mit bestimmten Qualitatseigenschaften. D a bislang gewerblich ver- triebene Leguminosenstarken in der Regel nur iiber Windsich- tung [l, 21 abgetrennt werden und damit die fur die anderen Starkearten bereits bestehenden Qualitatsanforderungen in der Bundesrepublik Deutschland [ 101 nicht erfullen konnen, muB zunachst ein praktikables Verfahren zur Starkeisolierung im LaboratoriumsmaRstab erarbeitet werden, um im Anschlurj daran qualitative Studien an diesen Starken moglich zu machen. Die vorliegende Arbeit berichtet uber Versuche, die Starkeabtrennung auf naBtechnischem Wege vorzunehmen.

2 Material und Methoden

Die Starkeabtrennungen wurden aufgrund ihres orientieren- den Charakters im Laboratoriumsmarjstab mit Erbsen bzw. Erbsenmehlen (vermahlen in einer Schlagkreuzmuhle) durch- gefuhrt.

416 starchklrke 39 (1987) Nr. 12. S. 416-421 8 VCH Verlagsgesellschaft mbH. D-6940 Weinheim. 1987 0038-9056/87/1212-0416$02.50/0