Crop Science 40:1482-1485 (2000)
© 2000 Crop Science Society of America
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Trypsin inhibitory activity measurement
Simplifications of the standard procedure used for pea seed
D. Pagea,
L. Quillienb and
G. Duca
a Institut National de la Recherche Agronomique, Unité de Recherche en Génétique et Amélioration des Plantes, 17 rue Sully, BV1540 F21034 Dijon Cedex, France
b Institut National de la Recherche Agronomique, Laboratoire de Biochimie et Technologie des Protéines, Rue de la Géraudière, BP 71627 F44316 Nantes Cedex 03, France
page{at}epoisses.inra.fr
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ABSTRACT
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A major breeding criterion for pea (Pisum sativum L.) is a low seed trypsin inhibitor activity (TIA), but the high cost of measurement limits the screening of new experimental lines. Our objectives were to devise two simplified procedures for screening for TIA using micro titration plates. In the first procedure, the inhibition induced by one single extract is measured. It allows for a rapid ranking of genotypes, but not calculation of seed TIA. Conversely, the second procedure is a titration procedure, where results are expressed in conventional trypsin inhibitor units (TIU). The time required for the measurements is reduced compared with that required with the standard procedures. The different methods were performed on cultivars ranging from low to high seed TIA. The results using the screening procedure showed very low standard deviations and allowed the ranking of the cultivars into three classes according to their seed TIA. The ranking was consistent with results obtained using the standard procedures. Results of the titration procedure were not significantly different from results obtained with the standard procedures on pea lines ranging from 2 to 10 TIU mg-1 dry weight.
Abbreviations: AFNOR, French Association for Normalization • AOCS, American Oil Chemists' Society • BAPA, benzoyl-DL-arginine-p-nitroaniline • I%, percentage of inhibition • TI, trypsin inhibitor • TIA, trypsin inhibitor activity • TIU, trypsin inhibitor unit
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INTRODUCTION
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ACCORDING TO THE ANIMAL FEEDING INDUSTRY, trypsin inhibitors (TI) are the major antinutritional factor of pea seed (Savage, 1989). The correlation between overall seed TIA and protein digestibility was negative for animals fed with pea, with TIA accounting for 50% of the lack of digestibility in piglets (Sus scrofa) (Le Guen, 1993). As a result, registration of cultivars having a high seed TIA has not been allowed in France since 1992. The limit is two TIA units higher than the two official control cultivars chosen each year by the registration committee. Therefore, any pea breeding program dedicated to the French market should consider low TIA a high-priority criterion.
The feed industry cares about the TIA of raw materials (seed batches) to adapt their incorporation into animal diets and needs a standard unit that allows comparison of different products (Kakade et al., 1974). Breeders need to accurately estimate the seed TIA of their new cultivars before registration, but more significantly, they need to easily screen experimental lines. The easier and cheaper the TIA measurement, the earlier breeders can screen their lines during breeding programs. Indeed, they generally screen simple traits in early generations to greatly reduce the number of experimental lines before studying more complex traits. Seed TIA seems to be simply inherited (Domoney et al., 1994) and could be screened for in early generations. Thus, the simplification of the screening procedure is an important economic objective.
Two reference procedures are available for screening, the American Oil Chemists' Society (AOCS) method (no. BA12 75 2983) and the French Association for Normalization (AFNOR) method (no. XP V18-202). The main difference between the two methods is the composition of the TI extraction buffer. The AOCS method uses an alkaline buffer, while the AFNOR method uses an acidic one. The other steps of both procedures are very similar. Trypsin inhibitor activity is determined by measuring the level of inhibition of a standard trypsin-substrate reaction resulting from the addition of different dilutions of a standard TI extract.
Neither of these procedures is adapted for routine use. First, they are two-step procedures. A pretitration is required to adapt the meal to the buffer ratio, which is then used to extract trypsin inhibitors. Second, the experimental conditions required to perform the test are very strict, such that calculation of standard TIU becomes impossible with a small variation of these conditions (Valdebouze et al., 1980). The AFNOR standard procedure was improved by measuring seed TIA in micro titration plates (Gaborit et al., 1993). However, this method still requires pretitration and very strict experimental conditions and therefore is not the simple method needed by pea breeders and users. Screening pea seed TIA still has a high cost, which is a limit for pea users and breeders.
Our objectives were (i) to devise two simple procedures for assaying TIA and (ii) to assess the accuracy of the results and evaluate whether pea users and breeders could adopt our procedures for their routine uses instead of the reference procedures.
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Materials and Methods
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Twenty cultivars and experimental lines were chosen from the INRA-Dijon pea germplasm collection to provide seed with TIAs ranging from 0.7 to 11.1 TIA units mg-1 (AFNOR method) (Table 1) . In addition, 200 F6 plants of a cross between `Frisson' (high TIA) and `Terese' (low TIA) were produced by single-seed descent. The two parents and their F6 progenies were sown during spring 1998 in a nursery at INRA-Dijon (0.5-m2 plots, 25 plants m-2). Seeds were harvested after maturation and ground on an ultracentrifuge mill, with maximum particle size of 0.2 mm.
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Table 1 Trypsin inhibitor activity (TIA) of 20 pea cultivars and INRA-Dijon experimental lines measured by the screening procedure, the titration procedure, and the French Association for Normalization (AFNOR) and American Oil Chemists' Society (AOCS) reference procedures
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The screening procedure was performed in 96-well micro titration plates. The first and the last columns of the plate were filled with 50 µL of water per well. No inhibitor was added, so these 16 wells constituted positive controls where 100% of the added trypsin reacts. For each sample, trypsin inhibitors were extracted by mixing 10 mg of ground material with 5 mL of 0.0025 M HCl, stirring constantly for 30 min. One-milliliter aliquots of this suspension were transferred into each of two 1.5-mL tubes and centrifuged 5 min at 30000 g at 4°C. Two 50-µL aliquots of the supernatant from each tube are transferred into two wells of a micro titration plate. Considering that four wells were used per sample (two 1-mL aliquots per sample and two wells per aliquot), the TIA of 20 flour samples could be assessed on one micro titration plate.
The following steps of the reaction were performed as previously described by Gaborit et al. (1993) except for minor modifications. Fifteen microliters of a trypsin solution, prepared as described by Kakade et al. (1974), were added to each well. After exactly 10 min, 125 µL of a solution of benzoyl-DL-arginine-p-nitroaniline (BAPA), prepared as described by Kakade et al. (1974), were added to each well. This BAPA is an artificial substrate that reveals the noninhibited trypsin as it becomes yellow when it reacts with the trypsin (Kakade et al., 1974). After exactly 30 min, the reaction was stopped by adding 25 µL of 30% (v/v) acetic acid. The absorbance of each well was then measured at 405 nm.
Percentages of inhibition (I%) were calculated by comparing the absorbance measured for each well to the mean value measured for the 16 positive controls of the plate
. The mean of the four subsamples measured for each flour sample is considered as the result for the sample.
The titration procedure was performed in 96-well micro titration plates. Trypsin inhibitor extract of a sample was prepared as follows. Fifteen milligrams of ground material were mixed with 5 mL of 0.0025 M HCl and stirred for 30 min. Suspensions were centrifuged 10 min at 30000 g at 4°C. Two measurements were performed for each sample. Each measurement used one 12-well row of a micro titration plate. The first well of the row was filled with 50 µL of water and constituted the positive control. The following well was filled with 50 µL of undiluted extract and was used as the reagent blank, as 25 µL of 30% acetic acid were immediately added into this well to inhibit the trypsin. The third well of the row was filled with 50 µL of undiluted extract and the nine remaining wells of the row were filled with 50-µL aliquots of each TI extract dilution prepared with 0.0025 M HCL following the dilution factors 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9. The theoretical quantity of ground sample (Qtheo) contained in a 50-µL aliquot of each respective dilution is 0.015, 0.03, 0.045, 0.06, 0.075, 0.09, 0.105, 0.12, and 0.135 mg.
Trypsin, BAPA, and acetic acid were then added to each well using the same procedure as described in the screening procedure except that the reaction was stopped by acetic acid after exactly 20 min. Absorbance was measured at 405 nm. The value of the reaction blank was then recalculated from values of all the assays containing the same pure or diluted TI extract. The reason for this correction is to eliminate any absorbance that is not due to an enzymatic degradation of the BAPA. Indeed, in this reaction blank, as trypsin is inhibited by acetic acid before adding the BAPA, if any absorbance is detected at 405 nm, it can only be due to some other components whose absorbance factor at 405 nm is not null. Corrected values were used to calculate the percentages of inhibition using the mean value of the eight positive controls of the plate.
One TIU is defined as an increase of 0.01 absorbance unit at 405 nm under standard conditions (Kakade et al., 1974). This means that one TIU corresponds with an increase of 1% of inhibition (relative to the positive control) per milligram dry weight. Therefore, the slope of the linear regression of percentages of inhibition against the theoretical quantity of ground material corresponds with the TIA per milligram of the fresh sample (TIAfw). This value was further corrected by the dry matter content of the sample (h%) to obtain the final TIA per milligram of dry sample (TIAdw): TIAdw = TIAfwh%. Only I% from 10 to 80 % were used for TIU calculations, because outside those limits the relationship between the theoretical quantity of ground sample and the absorbance of the assays is not linear and does not permit the measurement of TIA.
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Results and Discussion
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Percentages of inhibition obtained with the screening procedure ranged from 14 to 84 % (Table 1). Using the positive control as an internal reference eliminated most technical or environmental variations that may have occurred during measurements. Therefore results showed low standard deviations whatever the percentages of inhibition. For eight replicates, per sample standard deviations ranged from 0.32 to 1.43 % (Table 1).
The screening procedure allows ranking cultivars into different classes of TIA. Three classes could be arbitrarily defined according to results of the Student-Newman-Keuls test: (i) a low TIA group of cultivars from 14 to 20% of inhibition, (ii) an intermediate TIA group with cultivars from 23 to 60%, and (iii) a high TIA group with cultivars higher than 70% (Table 1). When TIA was higher than 8 TIU mg-1, discrimination between genotypes was not possible as inhibition reached a maximum of 84%, and such cultivars were classified as high TIA peas. The classification of all 20 cultivars and lines into three classes was consistent with results obtained with the AFNOR reference procedure. Rank correlation obtained with results of the two methods was highly significant (Table 1).
When the screening procedure was performed on F6 plants developed by single-seed descent from a cross between Frisson (high TIA) and Terese (low TIA), two populations were distinguished (Fig. 1) . One was centered around a low TIA value corresponding with the TIA of Terese, and the other was centered around a high TIA level quite similar to the TIA of Frisson. This bimodal distribution of the TIA of the F6 individuals was consistent with the hypothesis of Domoney et al. (1994) of one major locus controlling overall pea seed TIA. This trial shows that our assay is not mainly influenced by variations of experimental conditions and it can be used to describe the genetic control of seed TIA.
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Fig. 1 Distribution of seed trypsin inhibition activity (TIA) of pea F6 derived inbred lines from a cross between cultivars Terese (low TIA) and Frisson (high TIA). Inhibition corresponding with the two parents is indicated with arrows
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Our screening procedure is a powerful method to easily classify lines and to screen segregating populations. This procedure permits an easy comparison with reference samples of known TIA values, as they can be included on the same plate. In germplasm trials, for example, control cultivars can be included in each plate as internal references.
The titration procedure was developed to reduce the time needed for pea seed TIA titration by avoiding the pretitration of samples and simplifying preparation. We evaluated the consistency of the new measurement. Since this procedure eliminates all I% values higher than 80%, the higher the TIA of the sample the less the number of values remaining for the calculation of the linear regression slope and, consequently, the higher the variability of results. Indeed, a set of 15 measurements performed on `Aladin', whose mean TIA was 3 TIU mg-1, showed a standard deviation of only 0.1 TIU mg-1 (Table 1), whereas an equivalent set of 15 measurements on `Maro', whose TIA reached 8.7 TIU mg, had a standard deviation of 1 TIU mg-1. The titration procedure also gave consistent results for low or intermediate TIA peas, the high ones being sorted in the upper part of the TIA scale with a lower consistency of values.
We chose to improve the AFNOR reference method for the titration procedure by using the same acid buffer for TI extraction, as it allows for a better specific TI activity of the extract (Ferrasson, 1995). Moreover, because most pea cultivars exhibit seed TIA between 2 and 10 TIU mg-1, we adapted the titration experimental conditions for such samples. Thus, the titration procedure gave results consistent with those obtained with the AFNOR reference methods (Table 1). Conversely, a significant difference was observed between the AOCS procedure and the other procedures. This may be a consequence of the alkaline extraction of seed trypsin inhibitors used in the AOCS method. Protein extraction using alkaline buffer is less specific; hence, other components that interact with the coloration of the BAPA could be extracted.
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Conclusions
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The two procedures presented here simplify pea seed TIA measurements. The screening procedure allows comparisons of genotypes, and we propose it for use in genetic studies on pea seed TIA. The titration procedure allows comparisons of pea samples with other feed products or with pea samples from multi-location trials. The time required for pea titration using the titration procedure is reduced because no pretitration is required. The results obtained with both procedures are consistent with the AFNOR reference method.
Received for publication January 29, 1999.
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REFERENCES
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- Domoney C., Welham T., Ellis N., Hellens R. Inheritance of qualitative and quantitative trypsin inhibitor variants in Pisum. Theor. Appl. Genet. 1994;89:387-391.
- Ferrasson, E. 1995. Purification et caractérisation biochimique des inhibiteurs trypsiques de pois (Pisum sativum L.). Ph.D. thesis. Food Science, Univ. de Nantes, Nantes, France.
- Gaborit, T., L. Quillien, and J. Gueguen. 1993. Determination of trypsin inhibitors activity in seeds by a microtitre plate method . In A.V.d. Poel (ed.) International Workshop on Antinutritional Factors (ANFs) in legume seeds. Wageningen, the Netherlands. 1–3 Dec. 1993. EAAP, Wageningen, the Netherlands.
- Kakade M.L., Rackis J.J., McGhee J.E., Puski G. Determination of trypsin inhibitor activity of soy products: A collaborative analysis of an improved procedure. Cereal Chem. 1974;51:376-382.
- Le Guen, M.P. 1993. Pea proteins for piglets: Effects on digestive processes. Ph.D. thesis Animal Nutrition, Agricultural Univ., Wageningen, the Netherlands.
- Savage G.P. Antinutritive factors in peas. In: Huisman J., et al. , ed. Recent advances of research in antinutritional factors in legume seeds. Wageningen, the Netherlands: PUDOC, 1989:342-350.
- Valdebouze P., Bergeron E., Gaborit T., Delort-Laval J. Content and distribution of trypsin inhibitors and hemagglutinins in some legume seeds. Can. J. Plant Sci. 1980;60:695-701.
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ABSTRACT
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