storage-induced hardening in 20 common bean cultivars
TRANSCRIPT
STORAGE-INDUCED HARDENING IN 20 COMMON BEAN CULTIVARS
D. W. STANLEY,'.' T. E. MICHAELS,' L. C. PLHAK' and K. B. CALDWELL'
Accepted for publication February 21, 1990
ABSTRACT
Twenty cultivars of common bean (Phaseolus vulgaris L.) varying in color and size were grown in three consecutive growing seasons at diferent locations and then stored for 6 months at 30"C/85% RH to induce hardening. Bean hardness was measured instrumentally following soaking and cooking. All ail- tivars increased in hardness during storage by a factor ranging from I .S4 to 2.47. SigniJcant sources of variation in hardness included cultivar, environment and, largest, cultivar X environment interaction. Of the various chemical and physical tests conducted on beans before and after storage, those important in determining jinal hardness included fluorescence intensity (a predictor of a phenol polymerization-type reaction), phytate level, seed volume (larger cultivars hardened less) and water relationships (storage reduced the amount of bound water). The amount of water absorbed following storage was related to cultivar color, seed volume and hilum area but not seed coat thickness.
INTRODUCTION
The failure of reconstituted legume seeds to rehydrate and soften adequately during soaking and cooking processes results in lowered utilization of this im- portant source of protein in the developing world as well as increased fuel consumption. This defect, hard-to-cook (HTC), proceeds during storage and can be induced in the laboratory by elevated temperature and humidity conditions, paralleling the climate in tropical countries. All common beans (Phaseolus vul- garis L.) studied thus far harden, the extent of which is a function of time, temperature, humidity and cultivar interactions (Stanley and Aguilera 1985; Aguilera and Stanley 1985).
Few studies, however, have examined hardening for more than one growing season in multiple cultivars. Multiyear plantings are necessary in order to evaluate
'Department of Food Science 2Department of Crop Science University of Guelph Guelph. Ontario. Canada N l G 2W1 'To whom correspondence should be addressed.
Journal of Food Quality 13 (1990) 233-247. All Rights Reserved. 0 Copwight 1990 by Food & Nutrition Press, Inc . . Trirmbull. Connecticut. -733
234 D W STANLEY. T. E MICHAELS. L. C. PLHAK and K . B. CALDWELL
environmental effects; also, since breeding may be an effective control strategy for the HTC defect, multiple cultivar data are needed to estimate heritability. This paper presents the results from a study of 20 cultivars of common beans grown in three environments in which several potential hardening parameters were measured. A companion paper (Michaels and Stanley in preparation) will detail the genetic aspects of this work.
MATERIALS AND METHODS
Cultivars, Storage and Cooking
Twenty diverse cultivars of the common bean including 6 white or navy, 6 black, 2 red kidney, 2 red Mexican, 2 pinto and 2 Great Northern market types (Table I ) were selected in order to represent the genetic diversity of seed size and color characteristic of this species. The white beans were cultivars recom- mended for Ontario; others were developed in the United States, Central and South America. Beans were grown in three environments: in 1986 at the Elora Research Station of the University of Guelph, in the 1986-87 growing season of the Southern Hemisphere at the Department of Agronomy, University of Chile, Santiago, and in 1987 at the Woodstock Research Station of the University of Guelph. Similar standard agronomic practices were employed at all sites.
Following harvesting and field drying to a moisture level of approximately lo%, beans were transported to Guelph and stored in cloth bags under elevated temperature (30°C) and relative humidity (859) conditions to produce the HTC defect. Initially and following 6 months storage beans were soaked 18 h in distilled water at room temperature, drained and rinsed. Beans were then cooked in boiling distilled water for 1 h at a waterbean ratio of 5:l by volume, replacing water as necessary. The beans were then drained and held at room temperature until testing which took place at 21°C.
Testing Procedures
Hardness. Bean hardness was determined by either a compression procedure using a 30 g sample of whole cooked beans (Plliak et a/. 1987) or with a probe procedure using cotyledons from which the seed coat had been removed after cooking (Plhak et al . 1989). In addition, a portion of the separated seed coat was subjected to probe testing using the method of Stanley et a/ . (1989).
Water Relationships. Water absorption, water-holding capacity and moisture content were determined according to the methods of Plhak et a/. (1989).
Chemical Tests. Phenols were measured on an extract prepared by shaking 1 .O g ground whole beans and 25 mL methanol for 5 min by hand. The extract was then centrifuged 10 rnin in a clinical centrifuge at maximum speed. A 0.2
HARDENING IN BEAN CULTIVARS 235
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236 D. W . STANLEY. T. E MICHAELS. L. C. PLHAK and K. B . CALDWELL
mL portion of the supernatant was analyzed using the Folin-Ciocalteu reagent and compared to a standard curve prepared from tannic acid (Hincks and Stanley 1986). Fluorescence microscopy was used to measure the intensity of radiation emitted from the cut surface of uncooked bean cotyledons excited at 450-490 nm using the procedure of Stanley and Plhak (1989). An anion exchange method was used to recover phytate and a colorimetric procedure employed for phytate determination following an initial extraction with 2 . 4 7 ~ HC1 (Hincks and Stanley 1986). Nitrogen values were obtained using a macroKjeldah1 procedure.
Seed Characteristics. Seed volume was determined by weighing 100 seeds and assuming their density to be 1 .OO. Seed coat thickness and hilum area were calculated using light microscopy and a Zeiss Zidas image analysis system. Color of whole uncooked beans was estimated using reflectance data collected from a Hunterlab Model D25 colorimeter. 9 Y readings were selected to monitor light- ness after calibration of the instrument against a white standardized plate.
Agronomic Data. Bean yield and days to maturity data were recorded for each cultivar.
Statistical Analysis. ANOVA, correlation analysis and stepwise multiple regression analysis were performed using a Statistical Analysis System package (SAS Institute 1985). In stepwise multiple regression analysis variable selection was based on F - to enter and F - to remove probability levels of 0.10. Cluster analysis was performed using the BMDP-1M Program (Dixon 1983) with cor- relation coefficients being used as measures of similarity for grouping variables into clusters. Clusters were combined according to the minimum distance, or maximum similarity. of overall pairings of variables between two clusters. Table 2 lists the variables used in this study. their units of measure, sampling protocol, averages over all cultivars and standard deviations.
RESULTS AND DISCUSSION
Hardening Data
All cultivars studied hardened appreciably during storage. On average, stored beans required 5 16 N of force for compression/30 g cooked sample, 1.99 times that needed immediately postharvest (Tables 1, 2 ) . In previous work (Rivera et al . 1989) with a black seeded cultivar, beans with a compression force of greater than 400 Ni30 g or with a hardening ratio of greater than 2 were judged by a sensory panel to have unacceptable texture. These criteria, however, may not apply equally to the diverse group of bean market types in the present study.
Cultivar was a significant source of variation in final compression force, but not for initial force or the fina1:initial hardening ratio (Tables 1 , 3). The kidney beans Montcalm and Redkloud and the white bean Harofleet had significantly
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238 D W STANLEY. T . E. MICHAELS. L C. PLHAK and K B . CALDWELL
lower final compression forces than most other cultivars while the white bean Midland and black beans Midnight and Porillo 1 had the highest final compression forces. Final compression force could not be predicted accurately from the initial compression force ( r = .44; P > 0.05; 18 d o . Therefore, since the extent of hardening is independent of initial compression force, procedures aimed at de- tecting the propensity of beans to harden by measuring initial compression force have a low probability of success. Hardening ratio was correlated with initial compression force ( r = 0.84; P < 0.01; 18 d o . but not with final compression force (r = 0.07: P > 0.05; 18 d o .
A successful bean cultivar should not only have a low final compression value and low hardening ratio, but these characteristics should be consistent and in- dependent of where the beans are grown. All compression hardening parameters in this study were influenced by an interaction of cultivar with environment; cultivar x environment interactions were the greatest source of variation for the compression data (Table 3). Stability analyses of these data have been conducted and several cultivars identified for future breeding experiments.
Additional Analyses
Several additional analyses of quality were undertaken in the third season of the study with the aim of achieving a better understanding of the hardening process. One of these was a hardness test based on measuring the force required to puncture a cooked, dehulled bean. This procedure was not available until the second month of storage so that probe hardening ratio is a comparison of 2 and 6 month data. The lower average probe hardening ratio obtained (1.28) compared to the compression method ( 1.99) can be explained by the 2 month difference in period of comparison. Initial probe force was correlated with probe ratio ( r = 0.74; P < 0.01; 18 d o . but not with final probe force (r = 0.25; P > 0.05; 18 d o . Using season 3 data for all cultivars, no significant correlations were found between compression and probe methods for initial force, final force or hardening ratio values, which was not unexpected considering the different types of force involved and the contributions of seed coats to bean texture (Stanley et al. 1989).
Multivariate data analysis (cluster analysis) was used to determine the grouping of the large number of variables measured during the third season in this study with hardness parameters. Initial compression force was grouped with final water absorption. hilum area and phenol parameters ( P < 0.01) and nitrogen (P < 0.05) (Fig. 1). Final compression force was associated with a large cluster including initial compression force, phenol level, water absorption, seed and seed coat physical characteristics with a probability of approximately 0.05. Compression hardening ratio was grouped with color (P < 0.001), initial water- holding capacity and phytate ( P < 0.05). Initial probe force was associated with
HARDENING IN BEAN CULTIVARS 239
240 D W STANLEY.T E MICHAELS. L C PLHAKandK B CALDWELL
FINAL M)IIPRESSIOH FORCE
INITIAL COyPRESsKm FORCE
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FIG I CLL'STER ANALYSIS OF THIRD SEASON DATA Arrou s on abscissa indicate statistical significance ot cluster formation.
no other parameters. Final probe force was grouped with initial moisture content (P < 0.05). Probe hardening ratio was independent of other variables at the 5% level. The lowest amalgamation distance was greater than one, suggesting none of the variables were redundant.
In order to further explore the relationship among these measurements multiple stepwise linear regression of cultivar averages of the third season data was employed. using hardness parameters as the dependent variables. The results of these analyses (Table 4) are of interest both for what they indicate is of importance and what is not. Fifty to sixty percent of the total variation in the initial hardness parameters could be explained by the variables named. No common variables
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242 D W STANLEY. T E. M1CHAEL.S. L. C PLHAK and K B CALDWELL
were shared between the compression or probe methods for determining initial or final force. or their ratio. Although for all hardness parameters the coefficients of determination were statistically significant, R’ values for compression forces exceeded those for probe forces.
Stepwise regression identified seed volume as being associated with final compression force. Seed volume was also significantly and negatively correlated with final compression force (r = - .46; P S .05; 18 df) in that larger cultivars had lower values, and this was also one of the seed physical characteristics clustered with final compression force. The influence of seed volume could be associated with the seed coat since seed coat material would constitute a smaller percentage of the total sample as seed size increases, even taking into account the strong positive correlation ( r = .64; P G .01; 18 df) between seed volume and seed coat thickness. Further, seed coat volume expressed as a percentage of total seed volume for the third season data values (mean = 0.68%, range = 0.53 to 0.86%) was not significantly correlated with any force measurement. Observation of sample behavior in the compression cell showed that seed coats tended to form an enmeshed mat against the bottom of the cell that would resist the plunger force. Also, prior work has suggested that seed coats may harden during tropical storage via a phenol-fed polymerization-type mechanism (Stanley et 01. 1989) and this hardening contributes to the sensory properties of cooked beans.
The influence of fluorescence intensity and initial moisture on final probe force as identified by stepwise regression and cluster analysis, respectively, may be part of a similar process. It may be hypothesized that later maturing beans will have, as a result of less field drying, a higher initial moisture upon initiation of storage, and due to this higher moisture, respiration will initiate more rapidly than in drier beans. Phenol polymers would be expected to accumulate in higher levels in the beans that more rapidly initiate respiration, and these cultivars will exhibit greater fluorescence (Stanley and Plhak 1989), lower final water-holding capacity and a more pronounced HTC defect.
According to stepwise regression, large percentages of the variation in hard- ening ratio based on compression or probe methods was accounted for by initial force. This observation reinforced similar results from correlation and cluster analysis. Beans that had similar increases in compression force following storage had different hardening ratios if their initial compression forces differed. Higher initial force tended to result in a lower hardening ratio.
Initial compression force was strongly related to initial phenol content (r = .68; P S .001; 18 d o . Phenol determinations were made on whole beans so the data reflect phenols occurring mainly in the seed coat. The biological significance of this high correlation may result from the relationship between water imperme-
HARDENING IN BEAN CULTIVARS 243
244 D. W STANLEY.T E MICHAELS. L C P L H A K a n d K B C4LDWELL
ability of legume seed coats and high levels of phenolic compounds (Stanley and Aguilera 1985). Total phenols become less extractable during storage at tropical conditions (Table 2 : Plhak er al. 19871, perhaps because they become involved in a crosslinking reaction that inhibits water imbibition, restricts water uptake and reduces cell separation during cooking.
Although seed coat mechanical properties (force and deformation) were not mentioned in the regression equations of hardness parameters they did correlate significantly and negatively (r = - .51; - .53; P .05; 18 df) with final water absorption but not seed coat thickness. This observation can be interpreted as meaning that harder seed coats among stored cultivars present a barrier to water penetration into stored seeds during soaking and cooking, irrespective of seed coat thickness.
While moisture content and water absorption were significant contributors to initial compression force and the compression hardening ratio, respectively, water-holding capacity became important in predicting final compression force. This observation suggests that water is able to penetrate easily into the cotyledon cells initially but one consequence of storage-induced hardening is the inhibition of cellular water binding.
The only chemical test associated with final compression force by stepwise regression was phytate content (Table 4). Proctor and Watts (1987) reported no significant correlation between initial phytate content and initial cooking time of three white bean cultivars grown at three different locations. The importance of phytate to bean hardening has been investigated for about a half century but the relationship between the two is still not clear (Stanley and Aguilera 1985). In the present work i t might have been expected that lower levels of phytate measured after 6 months tropical storage would be associated with harder cul- tivars since this would indicate more phytase activity and less chelation of divalent cations that presumably migrate to the middle lamella and crosslink pectins, leading to a failure of cotyledon cells to separate during cooking. Fluorescence intensity was the only significant variable in the equation describing final probe force (Table 4). Since this parameter is thought to reflect polymerized phenols (Stanley and Plhak 1989). these results suggest that the hardening process is paralleled by an accumulation of phenolic material in the cell wall.
Total extractable phenols decreased an average of 41% during the storage period (Table 2 ) . Phenol breakdown products, along with free aromatic amino acids generated by protein hydrolysis (Hohlberg and Stanley 1987). could serve as the reactants in a polymerization-type reaction, perhaps catalyzed by perox- idase (Rivera cr al. 1989) and/or free radicals from membrane breakdown (Smith and Stanley 1989) that would result in reduced extractability. Recently, Srisuma et al. (1989) reported that the free phenolic acid content in seed coats and cotyledons of white beans was significantly higher in HTC than control samples.
HARDENING IN BEAN CULTIVARS 245
Water Relationships Because most of the variables identified as significant in bean texture were
either directly or indirectly related to water relationships, it became of interest to determine the nonhardening factors influencing these properties. Stepwise regression (Table 4) was used as before and it was found that for the three water relationship factors having significant equations bean color was the most im- portant independent variable. Even for final water-holding capacity, which had no significant variables in the equation, the independent nonhardening variable having the highest correlation was final phenol concentration (r = .40; P > 0.05; 18 df). This observation is important because the color parameter was very highly correlated with final phenol concentration (r = - .75; P G .001; 18 df) and approached significance with final phenol concentration (r = - .42; P > 0.05; 18 df). Also, it was noted that no significant correlations existed among the four water relationship parameters. Based on these data it is possible to conclude that darker cultivars with higher levels of phenols absorb more water but tend to bind less of it in the cellular structure.
Two further factors to be considered in water imbibition are, first, the strong relationship between final water absorption and hilum area (r = .73; P 6 .001; 18 df) and, second, the significant negative correlation between final water absorption and seed coat physical properties mentioned previously. Several struc- tural features (seed coat thickness, seed volume, hilum size) and a compositional factor (protein content) were reported to be important in water absorption of legumes (Stanley and Aguilera 1985); in the present work nitrogen level ap- proached significance (r = .44; P > .05; 18 df) when correlated to final water absorption. When beans stored at tropical conditions were scarified at the hilum before soaking water absorption was reduced, presumably since water trapped between the seed coat and cotyledons could then escape (Plhak et al. 1989). Deshpande and Cheryan (1 986) reported that hilum, micropyle and seed coat all played important roles in water uptake. Apparently, as the bean hardens the normal major route of water absorption-through the seed coat-is partially blocked and more is taken in through the hilum. Since water absorption decreased less than 10% during storage (Table 2) the hilum can successfully assume this role. Water-holding capacity, however, decreased 22% during storage (Table 2 ) and it is this loss of available water inside the cotyledon cell that prevents cell separation during cooking.
In conclusion, seed coat characteristics are not of great importance in dictating water relationships in beans since they do not control how much moisture enters the cotyledon cell. Phytate and phenol contents are cotyledon factors that can participate in chemical reactions resulting in restricted water binding and impaired cell separation during cooking. These findings support the multiple mechanism theory of bean hardening previously advanced (Hincks and Stanley 1986).
246 D. W. STANLEY. T. E. MICHAELS. L. C . PLHAK and K. B. CALDWELL
ACKNOWLEDGMENTS
This work was supported by the International Development Research Centre, the Ontario Bean Producers' Marketing Board, the Natural Sciences and Engi- neering Research Council and the Ontario Ministry of Agriculture and Food. The assistance of Robert Jackman, Michael Hincks, John McNeil, Gillian Ma- haffey and Xue Mei Wu is gratefully acknowledged.
REFERENCES
AGUILERA, J . M. and STANLEY, D. W. 1985. A review of textural defects in cooked reconstituted legumes-The influence of storage and processing. J . Food Proc. Preserv. 9, 145-169.
DESHPANDE, S. S. and CHERYAN, M . 1986. Microstructure and water uptake of Phaseolus and winged beans. J . Food Sci. 51, 1218-1223.
DIXON. W. J . 1983. BMDP Statistical Software Manual; University of Cali- fornia Press, Berkeley, California.
HINCKS, M. J . and STANLEY, D. W. 1986. Multiple mechanisms of bean hardening. J . Food Technol. 2 1 , 731-750.
HOHLBERG, A. I . and STANLEY, D. W. 1987. Hard-to-cook defect in black beans. Protein and starch considerations. J . Agric. Food Chem. 35, 571-576.
PLHAK, L. C . , STANLEY, D. W., HOHLBERG, A. I . and AGUILERA, J . M. 1987. Hard-to-cook defect in black beans-Effect of pretreatment and storage condition on extractable phenols and peroxidase activity. Can. Inst. Food Sci. Technol. J . 20, 378-382.
PLHAK, L. C.. CALDWELL, K . B. and STANLEY, D. W. 1989. Comparison of methods used to characterize water imbibition in hard-to-cook beans. J. Food Sci. 54, 326-329, 336.
PROCTOR, J . P. and WAITS, B. M. 1987. Effect of cultivar, growing location, moisture and phytate content on the cooking times of freshly harvested navy beans. Can. J . Plant Sci. 67. 923-926.
RIVERA, J . A . , HOHLBERG, A. I . , AGUILERA, J . M., PLHAK, L. C. and STANLEY, D. W. 1989. Hard-to-cook defect in black beans-Peroxidase characterization and effect of heat pretreatment and storage conditions on enzyme inactivation. Can. Inst. Food Sci. Technol. J . 22, 270-275.
SAS INSTITUTE INC. 1985. SAS Users' Guide: Sfufistics; SAS Institute Inc. Cary, North Carolina.
SMITH, J . L. and STANLEY, D. W. 1989. Nonenzymatic toughening of as- paragus: Identification of the phenol compounds involved and evidence for a free radical mediated mechanism. J . Food Biochem. 13, 271-287.
HARDENING IN BEAN CULTIVARS 241
SRISUMA, N., HAMMERSCHMIDT, R., UEBERSAX, M. A., RUENG- SAKULRACH, S . , BENNINK, M. R. and HOSFIELD, G. L. 1989. Storage induced changes of phenolic acids and the development of hard-to-cook in dry beans (Phaseolus vulgaris var. Seafarer). J. Food Sci. 54, 311-314, 318.
STANLEY, D. W. and AGUILERA, J. M. 1985. A review of textural defects in cooked reconstituted legumes-The influence of structure and composition. J . Food Biochem. 9, 277-323.
STANLEY, D. W. and PLHAK, L. C. 1989. Fluorescence intensity indicates bean hardening. J. Food Sci. 54, 1078-1079.
STANLEY, D. W., WU, X. and PLHAK, L. C. 1989. Seed coat effects in bean texture. J. Texture Stud. 20, 419-429.