Structural Characteristics of Stored Black Beans (Phaseolus Vulgaris L.)

Transcription

1 SCANNING VOL. 20, (1998) Received August 5, 1997 FAMS, Inc. Accepted with revision October 15, 1997 Structural Characteristics of Stored Black Beans (Phaseolus Vulgaris L.) JOSE DE J. BERRIOS, BARRY G. SWANSON,* W. ADELINE CHEONG* USDA, ARS, WRRC, Albany, California; *Department of Food Science & Human Nutrition, Washington State University, Pullman, Washington, USA Summary: Scanning electron microscopy (SEM) was used to study and compare the structural characteristics of black beans after 2 years of storage under ambient conditions (AC) and refrigerated hypobaric conditions (RHC). The exterior surface of the seed coats of the black beans were covered with small flake-like materials randomly clustered together. The hilar regions of black beans stored under either AC or RHC were similar, and the linea lucida exhibited well defined double palisade cell layers in both bean treatments. Cross sections of the seed coats revealed that the parenchymal cell layers of the beans stored under AC were disrupted and frequently exhibited large intercellular spaces between the cell layers. The parenchymal cell layers of the beans stored under RHC demonstrated little disruption of the cell layers, with only occasional intercellular spaces between the cell layers. Cotyledon cross sections of black beans showed that the cotyledon cells of the beans stored under RHC exhibited many large intercellular spaces characteristic of normal beans. Conversely, cotyledon cross sections of the beans stored under AC exhibited cotyledon cells with few small intercellular spaces characteristic of hard-to-cook beans. Key words: scanning electron microscopy, seed microstructure, black beans: Phaseolus vulgaris L., hard-to-cook, storage Introduction The common bean (Phaseolus vulgaris L.) is the main source of complementary protein in the cereal and starchy food diets of large segments of Mexico and Central and South American populations, as well as other regions in the world with subtropical and tropical climates. However, the development of the hardness defects of beans and other legumes stored for long periods of time at high temperatures and high relative humidities (Jones and Boulter 1983a, b; Mattson et al. 1950) negatively affect the overall quality of beans. Hardto-cook (HTC) beans are characterized by long cooking times Address for reprints: Jose De J. Berrios USDA, ARS, WRRC 800 Buchanan St. Albany, CA 94710, USA (Stanley and Aguilera 1985) and low nutritive values (Bressani et al. 1963). As a consequence, HTC beans yield poor quality foods, making them less acceptable to consumers, and can represent significant postharvest losses. The warm and humid conditions encountered in many regions of the world with subtropical and tropical climates accelerate the development of the HTC phenomenon in stored beans. Seed microstructure provides complementary information to physicochemical determinations. Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are used to study the microstructure of legume seeds. Scanning electron microscopy provides the food scientist with a necessary closer look at the dynamic structure of plant food tissues, as well as the physical and chemical changes in foods and food products during storage, processing, preparation, and packaging (Swanson et al. 1985). The susceptibility of beans to hardening is one reason for continuing research interest in bean microstructure (Garcia et al. 1993, Rockland and Jones 1974, Sefa-Dedeh and Stanley 1979, Silva and Luh 1978, Varriano-Marston and Jackson 1981). Storage conditions are essential for the preservation of bean quality. Therefore, by selecting the conditions used for beans storage, one may be able to preserve the fresh quality of beans. The objective of this study was to compare in situ the structural characteristics of black beans (Phaseolus vulgaris L.) after 2 years of storage under two selected conditions: Ambient and refrigerated hypobaric conditions. Materials and Methods Beans and Storage Conditions Black beans (Phaseolus vulgaris L.) cv. UI 906, grown in Idaho, were obtained from the Triangle Bean Co. (Homedale, Idaho). Black beans received 1 week after harvesting were divided into two groups. One group of beans was stored under ambient conditions (AC) of o C, 30 50% relative humidity (RH), at standard atmospheric pressure for 2 years, and the other group of beans was stored under refrigerated hypobaric conditions (RHC) of 4.5 o C, 50 60% RH, and atmospheric pressure of 125 mmhg for 2 years. Black beans stored under AC were maintained in a 22.7 kg kraft paper bag, while black beans stored under RHC were maintained in a reinforced 55 gal steel drum. The steel drum was

2 J. De J. Berrios et al.: Structural characteristics of stored black beans 411 equipped with a pressure tight lid fitted with a rubber gasket and a lucite viewing window. An inlet/outlet port with shutoff valves allowed the pressure inside the drum to be reduced to a selected pressure with the aid of a vacuum pump. For the purpose of this study, the internal pressure in the drum was reduced from standard atmospheric pressure of 760 mmhg to 125 mmhg to decrease the oxygen concentration to about 3.5%, in relation to 21% oxygen in the air at standard atmospheric pressure. Black beans were removed from storage after 2 years for preparation and study by SEM. Preparation of Beans for Scanning Electron Microscopy Black beans prepared for SEM observation were cleaned with dry cotton tissue to remove dirt and particles sticking to the surface. Clean beans were dried over CaSO 4 in a desiccator for 24 h. Prior to mounting, whole beans were flattened at one end with No. 600 sand paper, for the observation of the seed coat surface and the hilum. For examination of the transverse sections of the seed coats and cross sections of the cotyledons, the beans were fractured with a razor blade hit with a hammer. The whole and fractured bean cotyledons were directly mounted to stabs using double-sided adhesive tape, without any previous fixation treatment. Colloidal graphite cement (Ted Pella) was applied around the bottom of the bean cotyledons to provide better specimen conductivity. The mounted beans cotyledons were coated with approximately 30 nm of gold using a Technics Hummer V sputter coater. The beans were examined using a Hitachi S570 microscope operating at 20 kv, and micrographs were recorded with Polaroid Type 55 (P/N) film. face deposits observed by Hughes and Swanson (1985) on black bean seed coat, were present. The significance of deposits on the surface of the seed coats to processing of beans is not reported, but large quantities of deposits on the seed coat surfaces may have negative effect on the luster and acceptability of beans. Hilum: Whole and Cross Section Scanning electron microscopy observations of the hilum and hilar region of black beans stored at AC and under RHC were similar. The external structures of the hilum of black beans observed at low magnification (Fig. 3) exhibit a spongylike amorphous microstructure similar to the hila of black gram and Carioca beans examined by Joseph et al. (1993) and Garcia et al. (1993), respectively. The hilum of black beans observed at high magnification (Fig. 4) reveals the presence of cavities arranged in a honeycomb-like structure or a crosslinking network that appears to protrude inside the hilum. The presence of cavities in the structure of the hilum provides an obvious site for water penetration into the bean. Kyle and Randall (1963) studied water entry at the hilum, micropyle, and raphe in Great Northern and Mexican red market class cultivars of common beans Phaseolus vulgaris L. For Great Northern beans, the micropyle was the site of greatest water entry. For Mexican red beans, the raphe was the most important site of water entry, with the hilum being the next most important and the micropyle the least important. Korban et al. (1981) studied cultivars of pinto beans and concluded that the raphe or the hilum was the primary site Results and Discussion Seed Coat Surface The surface of the seed coats of black beans (Figs. 1, 2), exhibits a distinctive and visible pattern or fingerprint similar to that described by Hughes and Swanson (1985) in another cultivar of black beans. Garcia et al. (1993) also observed similar fingerprint patterns on the surface of the seed coats of Carioca beans. In this study, the surface of the seed coats of black beans stored at AC and under RHC was similar (Fig. 1). Garcia et al. (1993) also reported that the surface of the seed coats of normal and HTC Carioca beans was similar, indicating that the hardening process in beans does not affect the external appearance of the seed coat. The surface of the seed coats of black beans stored at AC and under RHC was covered with small flake-like materials randomly clustered together at low magnification. The flake-like materials that cover the seed coat surfaces of black beans could be seen as larger, smoother, and more swollen at greater magnification (Fig. 2) than in Figure 1. The seed coat surfaces of black beans (Figs. 1, 2) were free of pits, although scattered deposits, similar to the sur- FIG. 1 Seed coat surface of black bean.

3 412 Scanning Vol. 20, 5 (1998) FIG. 2 Seed coat surface of black bean at higher magnification. of water uptake. Agbo et al. (1987) studied the water imbibition of a cultivar of black beans ( San Fernando ) and two cultivars of white beans ( Sanilac and Nep-2 ) and found that the black bean cultivar had a higher resistance to water uptake than the two white bean cultivars, due to the presense of an occluded micropyle. The cross sections of the hilar region of black beans (Figs. 5, 6) demonstrate that the single layer of epidermal palisade cells in the seed coats of black beans extends into the hilar region as a double layer of palisade cells. The double layer of palisade cells observed in lentil seeds was believed to be an optical effect called the linea lucida, which gave the optical impression that the palisade cell layer in lentil seeds consisted of two distinct cell layers (Hughes and Swanson 1986). However, our study of black beans reveals that the linea lucida is actually a well defined structure denoting two layers of palisade cells, as was also observed by Agbo et al. (1987) in black and white bean cultivars, by Chilukuri and Swanson (1991) in adzuki and Mexican red beans, and by Joseph et al. (1993) in cultivars of black, green, and red gram. No such double palisade layer was observed in a cultivar of black beans by Chilukuri and Swanson (1991). Therefore, the presence or absence of a double palisade layer may be characteristic of bean cultivars. Since the hilum is a site for water entry into the bean (Kyle and Randall 1963, Varriano-Marston and Jackson 1981), the structure of the hilar region may influence the rate and quantity of water imbibed by the beans. The tracheid bar and the vascular tissue were observed in FIG. 3 External structures of black bean hilum. FIG. 4 External structures of black bean hilum. Cavities are observed as honeycomb-like structure.

4 J. De J. Berrios et al.: Structural characteristics of stored black beans 413 the subhilar region of black beans (Fig. 6). Tracheid bars and vascular tissues were also observed in lupinus texensis cultivars by Bragg (1983), and in black, green, and red gram by Joseph et al. (1993). The tracheid bar and vascular tissue are structures associated to aid and/or control the water entry into the seed (Weier et al. 1974) and are also used for cultivar identification (Bragg 1983). No subhilar plug was observed in black beans in this study or previously reported for other black bean cultivar by Chilukuri and Swanson (1991). However, a plug was observed in the subhilar regions of black, green, and red gram (Joseph et al. 1993) and in cultivars of adzuki and Mexican red beans (Chilukuri and Swanson 1991). The funiculus (Fig. 6) is a stalk of the ovule containing vascular tissue, partially collapsed during the fracturing process. Seed Coat Cross Sections The cross sections of the black bean seed coats (Figs. 7 10) exhibit the three characteristic and distinctive cell layers of the common beans (Phaseolus vulgaris L.), the palisade cell, subepidermal pillar (hourglass) cell, and parenchyma cell layers (Garcia et al. 1993, Sefa-Dedeh and Stanley 1979, Swanson et al. 1985). At low magnification, no apparent differences were observed when comparing the palisade and subepidermal pillar cell layers of black beans stored under AC versus RHC. Nevertheless, the parenchyma cell layers, making up the innermost portion of the seed coat closest to the cotyledon, were disrupted in beans stored under AC (Fig. 7) and frequently exhibited large intercellular spaces between the cell layers, while the parenchyma cell layers of the beans stored under RHC (Fig. 8) exhibited a consistent set of overlaid layers randomly organized and with occasional intercellular spaces between the cell layers, above the cotyledon structure. The palisade and subepidermal pillar cell layers of black beans stored under AC and RHC are presented at higher magnification in Figures 9 and 10. The palisade cell layer of black beans stored under AC and RHC was similar. However, the subepidermal pillar cell layer of beans stored under AC and RHC demonstrated some visible differences. The subepidermal pillar cell layer of beans stored under AC (Fig. 9) was partially devoid of observed mucilaginous substances, possibly of pectinaceous and glucanaceous origin. Conversely, the subepidermal pillar cell layer of beans stored under RHC (Fig. 10) was covered by the mucilaginous substances which may aid in the hydration of the beans during soaking. Water absorption by a cultivar of black beans ( San Fernando ) and two cultivars of white beans ( Sanilac and Nep-2 ) resulted in an observable swelling of the palisade, sub-epidermal pillar and parenchyma cell layers (Swanson et al. 1985). Similar observation was previously reported by Saio et al. (1973) for soybean. Crystal-like structures are observed in the subepidermal FIGS. 5 AND 6 Cross section of black bean hilum region, seed coat, and cotyledon. The seedcoat (SC) showing the single layer of epidermal palisade cells, the double palisade (DP) layers of cells at the hilum; the tracheid bars (T) and vascular tissue (VT) in the subhilum region; remnants of the funiculi (Fn) are visible. The cotyledons (C) and cotyledonary fissure (F) are also observed.

5 414 Scanning Vol. 20, 5 (1998) pillar cell layer of black beans stored under AC (Fig. 9). Calcium oxalate crystals were observed in the seed coat structures of legume seeds (Grimson et al. 1982, Webb and Arnott 1982). Mineralized structures may be associated with seed hardening, since higher concentrations of lignin, calcium, and silica in the palisade cell layer are reported for HTC legume seeds compared with normal legume seeds (Rodriguez and Mendoza 1990, Saio 1976, Sefa-Dedeh and Stanley 1979). Cotyledon Cross Sections Cotyledon cross sections of black beans (Figs. 11, 12) exhibit storage cells containing starch granules embedded in a protein matrix. Scanning electron microscopy examination of black (Hughes and Swanson 1985), adzuki, and other bean cotyledon cells (Joseph et al. 1993, Sefa-Dedeh and Stanley 1979), exhibited large µm spherical starch granules embedded in a protein matrix. Small 1 10 µm spherical to oval shaped individual protein bodies generally comprised the protein matrix of black beans (Hughes and Swanson 1985). The cotyledon cross sections of black beans stored under AC (Fig. 11) exhibit cotyledon cells with few small intercellular spaces and large adhesion areas between cotyledon cells. Conversely, the cotyledon cells of black beans stored under RHC (Fig. 12) exhibit many large intercellular spaces and small adhesion areas between cotyledon cells. The large intercellular spaces and small adhesion areas observed between cotyledon cells in beans stored under RHC may partially explain the faster initial water imbibition and subsequent shorter cooking time exhibited by the beans stored at RHC when compared with the beans stored under AC (Fig. 13, Table I). Large intercellular spaces and small adhesion areas between cotyledon cells were observed in normal beans by Hohlberg and Stanley (1987), Paredes-Lopez et al. (1989), and Garcia et al. (1993). On the other hand, small intercellular spaces and large adhesion areas between cotyledon cells were observed in HTC beans by Jones and Boulter (1983b) and Hincks and Stanley (1987). Jones and Boulter (1983b) postulated that the strong adhesion between cotyledon cells of HTC beans was a consequense of the formation of insoluble Ca and Mg pectate in the middle lamella, while Hincks and Stanley (1987) postulated that it was a consequence of lignification of the middle lamella. The results of the above studies indicate that beans exhibiting many large intercellular spaces and small adhesion areas between cotyledon cells are associated with normal beans, while beans exhibiting few small intercellular spaces and large adhesion areas between cotyledon cells are associated with HTC beans. The cytoplasm and plasmalemma of black beans stored at AC and RHC (Figs. 11, 12) occupy the total cotyledon cell volume. However, Garcia et al. (1993) reported that although the cytoplasm and plasmalemma of normal Carioca beans occupied the total cotyledon cell volume, HTC Carioca beans exhibited an accentuated contraction of the cytoplasm and plasmalemma separated from the cell walls. No chemical fixation was used to prepare black beans for SEM in this study, while Garcia et al. (1993) used glutaraldehyde/ formaldehyde fixation for preparation of Carioca beans for SEM. Hughes and Swanson (1985) observed the structural characteristics of normal black beans with and without FIGS. 7 AND 8 Cross sections of black bean seed coats of beans stored at ambient (Fig. 7) and refrigerated hypobaric conditions (Fig. 8), exhibiting the palisade (Pl), subepidermal pillar cells (PC), and parenchyma (Pr) cell layers characteristic of legume seeds.

6 J. De J. Berrios et al.: Structural characteristics of stored black beans 415 FIGS. 9 AND 10 Cross section of black bean seed coats of beans stored at ambient (Fig. 9) and refrigerated hypobaric conditions (Fig. 10) at higher magnification, exhibiting the palisade (Pl) and subepidermal pillar cells (PC). Part of the parenchyma (Pr) cell layers are also observed. FIGS. 11 AND 12 Cross section of black bean cotyledon cells of beans stored at ambient (Fig. 11) and refrigerated hypobaric conditions (Fig. 12). Elliptical starch granules (S), protein matrix (PM), cell walls (CW), and intercellular spaces (I) are noted.

7 416 Scanning Vol. 20, 5 (1998) Water absorption coefficient (g water/100 g dry beans) TABLE I Time (hr) of hydration Cooking time (min) a of black beans Soaked b Year AC RHC 0 c 29.7 ± ± ± ± ± ± ± 2.7 a Cooking times in min of last bean cooked to 100% in bean cooker (Burr et al. 1968). b Beans soaked in distilled water for 12 h at ambient conditions prior to cooking. c Beans cooking test done prior to storage. Storage conditions: AC= room conditions of o C and 30 50% RH. RHC= refrigerated hypobaric conditions of 4.5 o C, 50 60% RH, and atmospheric pressure of 125 mmhg. Values represent means of triplicate analyses. glutaraldehyde/formaldehyde fixation with SEM. Scanning electron micrographs of normal black beans without glutaraldehyde/formaldehyde fixation exhibited cytoplasm and plasmalemma occupying the total cotyledon cell volume. However, when normal black beans were fixed in glutaraldehyde/formaldehyde solutions, cytoplasm and plasmalemma were separated from the cell walls (Hughes and Swanson 1985). For SEM, the best specimen preparation is the least preparation (McAlear 1972). Therefore, glutaraldehyde/formaldehyde fixation may account for the differences or artifacts in cotyledon cell contents and arrangements observed between stored black beans of the present study and Carioca beans of the Garcia et al. (1993) study; or between stored black beans of the Hughes and Swanson (1985) study and Carioca beans of the Garcia et al. (1993) study. AC RHC FIG. 13 Water absorption coefficient of black beans stored under ambient (AC) and refrigerated hypobaris conditions (RHC) for 2 years. 24 Conclusions Scanning electron microscopy is an important tool for studying seed microstructure. It provided excellent information regarding the changes in structural characteristics that occurred in beans stored under ambient versus refrigerated hypobaric conditions. In addition, SEM allowed us the flexibility for observing the specimens in their natural state without the use of fixatives, which provided minimum manipulation of the specimen with subsequently less chance of introducing image artifacts. The difference in structural characteristics observed in beans stored under ambient and refrigerated hypobaric conditions will be further evaluated in a subsequent study of the physicochemical characterization of the stored beans. References Agbo GN, Hosfield GL, Uebersax MA, Klomparens K: Seed microstructure and its relationship to water uptake in isogenic lines and a cultivar of dry beans (Phaseolus vulgaris L.). Food Microstruct 6, (1987) Bragg LH: Seed coats of some lupinus species. Scan Electron Microstruct IV, (1983) Bressani R, Elias LG, Valiente AT: Effect of cooking and of amino acid supplementation on the nutritive value of black beans (Phaseolus vulgaris). Br J Nutr 17, (1963) Burr HK, Kon S, Morris HJ: Cooking rates of dry beans as influenced by moisture content and temperature and time of storage. Food Technol 22, (1968) Chilukuri A, Swanson BG: Microstructure of adzuki beans (Vigna angularis cv. Express). Food Struct 10, (1991) Garcia E, Lajolo FM, Swanson BG: A comparative study of normal and hard-to-cook brazilian common beans (Phaseolus vulgaris): Ultrastructural and histochemical aspects. Food Struct 12, (1993) Grimson MJ, Arnott HJ, Webb MA: A scanning electron microscopy study of winged twin crystal in the bean legume. Scan Electron Microstruct III, (1982) Hincks MJ, Stanley DW: Lignification: Evidence for a role in hard-tocook beans. J Food Biochem 11, (1987) Hohlberg AI, Stanley DW: Hard-to-cook defect in black beans. Protein and starch consideration. J Agric Food Chem 35, (1987) Hughes JS, Swanson BG. Microstructural changes in maturing seeds of the common bean (Phaseolus vulgaris L.). Food Microstruc 4, (1985) Hughes JS, Swanson BG: Microstructure of lentil seeds (Lens culinaris). Food Microstruct 5, (1986) Jones PMB, Boulter D: The cause of reduced cooking rate in Phaseolus vulgaris following adverse storage conditions. J Food Sci 48, , 649 (1983a) Jones PMB, Boulter D: The analysis of development of hardbean during storage of black beans (Phaseolus vulgaris L.). Qual Plant Plant Foods Hum Nutr 33, (1983b) Joseph E, Crites SG, Swanson BG: Microstructure of black, green, and red gram. Food Struct 12, (1993) Korban SS, Coyne DP, Weihing JL: Rate of water uptake and sites of water entry in seeds of different cultivars of dry bean. Hort Sci 16, (1981) Kyle JH, Randall TE: A new concept of the hardseed character in Phaseolus vulgaris L, and its use in breeding and inheritance studies. Am Soc Hort Sci 83, (1963)

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