Seed coat phytochemistry of both resistant and susceptible seeds affords some protection against the granivorous beetle Callosobruchus maculatus

The seed coat lies at the interface between the internal structures of the seed and the external environment and thus represents a key arena in the study of seed-herbivore interactions. Callosobruchus maculatus is a cosmopolitan pest of legume seeds, and under post-harvest conditions, females interact directly with the seed testa prior to laying their eggs. Here we investigate the effect of chemical extracts of the seed coat of the resistant Phaseolus vulgaris and the susceptible Vigna unguiculata beans on egg laying preferences and larval development of C. maculatus. Seed coat extracts contained phenolic, glycoside and alkaloid compounds. Upon re-incorporation of extracts into artificial host beans it was found that that several seed coat extracts from both the resistant and susceptible varieties reduced female oviposition and disrupted larval growth and development. However, none of the extracts assayed resulted in complete ovipositional or developmental failure suggesting that complete resistance in P. vulgaris is derived from other physical or chemical properties of the seed and/or seed coat that function either alone or synergistically. Further work is required to elucidate the importance of synergistic interactions between different physiological defence mechanisms on overall plant (seed) resistance.     

Identification,biochemical characterization and biological role of defense proteins from common bean genotypes seeds in response to Callosobruchus maculatus infestation

Common bean is a legume of significant socioeconomic importance and is cultivated worldwide. This crop is affected by several pests and diseases, which cause considerable economic losses and reduce yield. In recent years, several studies have demonstrated the role of proteins and peptides with activity against a wide range of insects and pathogens. The objective of this work was to identify defense proteins, such as antimicrobial peptides, protease and amylases inhibitors in common bean genotypes and evaluate the relationship of these proteins with Phaseolus vulgaris seed resistance to Callosobruchus maculatus infestation. Nineteen common bean genotypes were subjected to protein extraction, pH 5.4, and precipitation with ammonium sulfate at 70% saturation. The obtained extracts were separated by tricine gel electrophoresis. Experiments were carried out with natural seeds of common beans and artificial seeds (Vigna unguiculata seeds covered with seed coats of common beans) to evaluate the rate of oviposition and development of the insect species Callosobruchus maculatus. Lipid-transfer proteins were identified in nine genotypes whereas defensins were present in all genotypes. The inhibitory activity of α-amylases and trypsin and fungal development were determined in crude extracts (50 μg mL⁻¹). The results also indicated that the extracts from all bean genotypes inhibited the activity of human salivary α-amylase and C. maculatus larval α-amylase. Except for the extracts of four genotypes, all other extracts inhibited trypsin activity. None of the extracts from the evaluated bean genotypes inhibited the growth of tested fungi. Natural seeds from all genotypes did not inhibit insect oviposition, however, the larvae did not survive after feeding on these seeds. Artificial seeds containing seed coat flour of all genotypes inhibited the oviposition of C. maculatus, indicating that the seed coat was also repellent to insect.     

Rice bean (Vigna umbellata Thunb. Ohwi and Ohashi) protection against Callosobruchus maculatus F.” by presence of protein profile

To identify defence responses of rice bean against Callosobruchus maculatus, 10 genotypes of rice bean and a mungbean variety, PAU 911 as susceptible check were evaluated. The rice bean genotypes were found to restrain the growth and development of C. maculatus at different larval stages. To justify these results, seeds of different test genotypes were examined for the presence of plant protease inhibitors which inhibit insect proteases causing reduction in the availability of amino acids necessary for their growth and development. Trypsin, chymotrypsin and cysteine protease inhibitor content was significantly higher in rice bean genotypes which ranged from 299.99 to 342.97 TIU g⁻¹ seeds, 235.07 to 251.45 CIU/g seeds and 1060.70 to 1574.99 CPIU/g seeds, respectively as compared to mungbean variety, PAU 911 (235.52 TIU/g seeds, 217.91 CIU/g seeds and 499.99 CPIU/g seeds, respectively). The protein profile showed a polypeptide band of 25–30 kDa corresponding to presence of protease inhibitors in rice bean genotypes except PAU 911. The results indicate that protease inhibitors in different genotypes of rice bean are very effective against C. maculatus.     

Tryptic fragments of phaseolin from protein isolates of Phaseolus beans

Crystalline and amorphous protein isolates from white kidney and navy beans (Phaseolus vulgaris) and baby lima and large lima beans (Phaseolus lunatus) were subjected to tryptic hydrolysis. The hydrolysis was monitored by the nitrogen solubility index (NSI) and reverse phase-high performance liquid chromatography/mass spectrometry (RP-HPLC/MS) analysis of the hydrolysates. The results of the NSI and RP-HPLC showed that the tryptic fragments of the crystalline isolates possessed higher solubility properties than the fragments of the amorphous isolates. The results of the MS study of phaseolin peptides in the RP-HPLC fractions of the hydrolysates showed that trypsin-specific bonds located in β-structured regions of β-phaseolin are resistant to tryptic hydrolysis, and that the most susceptible bonds to trypsin hydrolysis are located in regions not directly involved in the formation of secondary structural units, but in regions of disordered structure, or in regions interconnecting secondary structural units. Some trypsin-specific bonds located within α-helical structures were also cleaved by trypsin hydrolysis.     

Influence of germination on the nutritional quality of Phaseolus vulgaris and Cajanus cajan

Germination is a method that can modify the presence of nutrients and antinutrients in legume seeds. In this study, the changes in trypsin inhibitors activity (TIA), phytic acid, tannins, ascorbic acid, thiamine, protein digestibility and minerals in germinated black beans (Phaseolus vulgaris L.), white beans (Phaseolus vulgaris L.) and pigeon beans (Cajanus cajan L. Millsp) were evaluated. The ungerminated grains were analysed as control. A significant decrease in the content of TIA of pigeon beans (19.2%), white beans (52.5%) and black beans (25%) was observed. The reduction of phytic acid was more than 40% for the three grains germinated; for the tannins, the reduction was of 14.3% for pigeon beans, 19% for black beans and 36.2% for white beans. Germination increased the protein digestibility in a 2-4% range, and also increased the ascorbic acid by 300% for white beans, by 33% for black beans and by 208% for pigeon beans. The thiamine content increased more than 26.7%. Germination affected the mineral content erratically, depending more on the grain and the type of mineral. These variations in the content of nutrients and antinutrients of the germinated grains are attributed to the joint effect of the germination and previous soaking the grains were subjected to.     

Variation in the response of Callosobruchus maculatus (F.) to a resistant veriety of cowpea

A Nigerian cultivar of the cowpea has previously been identified as being less suitable than conventional varieties for the development of a local strain of C. maculatus (F.). The survival and duration of development of the immature stages of three strains of C. maculatus in the dried seeds of this cultivar were compared with the performance of the same strains of beetle on a susceptible variety. In all beetle strains, fewer larvae survived to produce adults, and the duration of development was significantly extended, in the resistant cowpea cultivar. A Brazilian strain produced fewest adults and a Nigerian strain had the longest mean development period. Combining all the data, it appeared that a strain from the Yemen Arab Republic was least influenced and that from Brazil most adversely affected by the resistant cowpea. Evidence is presented indicating that factors in both the cowpea seeds and the beetles themselves may contribute to the poorer performance of bruchids in the resistant as compared to the susceptible variety.     

Effect of cooking on the composition of beans (Phaseolus vulgaris L.) and chickpeas (Cicer arietinum L.)

The effect of cooking on levels of nutrients and anti-nutritional factors in beans and chickpeas was investigated. Significant (p < 0.05) variation existed among the beans and chickpeas with respect to their crude protein, starch, soluble dietary fiber (SDF), insoluble dietary fiber (IDF), total dietary fiber (TDF), resistant starch (RS), trypsin inhibitor activity (TIA), mineral, phytic acid, tannin, sucrose and oligosaccharide (raffinose, stachyose and verbascose) contents. Cooking beans and chickpeas in water significantly increased protein, starch, SDF, IDF, TDF, Mn and P contents (on a dry weight basis), whereas reduced ash, K, Mg, TIA, tannin, sucrose and oligosaccharide contents were observed. Colored beans (black, cranberry, dark red kidney, pinto and small red bean) contained tannins, whereas little tannin in white-colored beans (great northern and white pea bean) and chickpeas (Desi and Kabuli type) was detected.     

Fitness in a malathion resistant Tribolium castaneum strain; feeding,growth and digestion

Extensive historical use of malathion in grain stores has led to the universal development of resistant strains of the red flour beetle, Tribolium castaneum Herbst. Adaptation to insecticide stress often reduces general fitness. To investigate non-defensive attributes physiological and biochemical properties (that may underlie fitness) of a field-derived malathion-resistant strain (larval resistance factor of 490) were measured against those of a susceptible strain and also with susceptible T. confusum. Further inhibition in vitro of gut proteolysis was determined for the resistant and susceptible strains of T. castaneum.The weights of whole gut tracts of the susceptible T. castaneum strain and T. confusum were similar but the resistant strain had significantly lower weight and width of whole gut tract. The gut of the resistant strain had more, and more intense, electrophoretic protein staining bands. Food consumed through the life cycle was significantly less for the resistant strain. Inhibitors for cysteine proteases had less effect in vitro in the resistant strain than in the susceptible strain. An inhibitor for serine proteases had almost no effect on proteolysis in the resistant strain.The reduced gut dimensions and food consumed, together with lower larval protein, appear to indicate a fitness cost that may be associated with malathion resistance. However, this may be offset by enhanced gut proteins and a relative greater protein content of the adult stage. The latter may be related to the reported increased reproductive capacity of the resistant adult and consequent superior fitness, compared to the susceptible. The lowered response of the resistant strain to cysteine and serine protease inhibitors may interact with adaptations to dietary inhibition in cases where resistant strains need managing, if this result is confirmed to be widespread.     

Evaluating the combination of a parasitoid and a predator for biological control of seed beetles (Chrysomelidae: Bruchinae) in stored beans

The two seed beetle species Acanthoscelides obtectus and Callosobruchus maculatus are among the economically most important pests of stored legume seeds world-wide. Insect natural enemies for biological control were considered in IPM strategies to control bean weevils in store. In this contribution, the control effect by a combination of the parasitoid Anisopteromalus calandrae and the predator Xylocoris flavipes on both seed beetle species was evaluated in a laboratory study on black eyed beans Vigna unguiculata. The combination of parasitoid and predator was most effective against both species of seed beetles. However, in C. maculatus the combination was not significantly more effective compared to A. calandrae alone, suggesting a poor effect on adults and no effect on eggs and larvae by X. flavipes. In the case of A. obtectus, the suppressive effect by the combination of the parasitoid and the predator was high. For C. maculatus, germination of black eyed beans was approximately three times higher when both natural enemies were present. The combination of A. calandrae and X. flavipes is considered a promising component for integrated control of A. obtectus.     

Bioactivity of selected plant-derived essential oils against Zabrotes subfasciatus (Coleoptera: Bruchidae)

The common bean Phaseolus vulgaris L. (Fabaceae) is an important vegetable protein source and constitutes a significant part of the diet in many tropical countries. The Mexican bean weevil Zabrotes subfasciatus (Boheman) (Coleoptera: Bruchidae) is one of the main pests affecting stored beans: it causes extensive qualitative and quantitative grain loss. We investigated the bioactivity of the essential oils extracted from Chenopodium ambrosioides L. (CA-EO), Ocimum gratissimum L. (OG-EO), and Schinus terebinthifolius Raddi (ST-EO) against Z. subfasciatus. After 12-h treatment, CA-EO and OG-EO at 20.0 μL/L of air killed 100% Z. subfasciatus, whereas ST-EO at 100.0 μL/L of air afforded 100% Z. subfasciatus mortality after 24 h. CA-EO provided the lowest 24 h LD₅₀ (0.8 μL/L of air) and displayed efficient repellent activity against Z. subfasciatus. Our results demonstrate that CA-EO is a potentially economical and environmentally friendly alternative to manage Z. subfasciatus in stored beans.     

Effect of legume seed extracts on the inhibition of proteolytic activity and muscle degradation of fresh water prawn (Macrobrachiumrosenbergii)

Trypsin inhibitors in the extracts from soybean (Glycine max), adzuki bean (Vigna angularis), bambara groundnut (Vigna subterranea) and red kidney bean (Phaseoulus vulgaris) varied in amount and molecular weight. The soybean extract had the highest level of trypsin inhibitor with molecular weight (MW) of 21 kDa, followed by bambara groundnut extract possessing trypsin inhibitor with MW of 15 kDa. Both extracts showed a more effective inhibition towards crude protease extract (CE) from the hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) than the extracts from adzuki and red kidney beans. Activity staining also reconfirmed the higher inhibitory activity on CE from hepatopancreas by the extracts from both soybean and bambara groundnut. The extracts from all seeds were able to inhibit the degradation of fresh water prawn meat containing CE in a concentration dependent manner. Based on inhibitor study, the extracts from soybean and bambara groundnut can be a potential aid to suppress the muscle softening of fresh water prawn, mediated by trypsin-like proteases released from hepatopancreas, during extended iced storage.     

Trypsin Inhibitor from 3 Legume Seeds: Fractionation and Proteolytic Inhibition Study

ABSTRACT: The trypsin inhibitor from navy beans (Phaseoulus vulgaris), red kidney beans (Phaseoulus vulgaris L.), and adzuki beans (Vigna angularis) provided by the Royal Project Foundation in Thailand was isolated by heat and ammonium sulfate (AS) precipitation. Incubation at 70 °C for 10 min produced the highest trypsin inhibitor recovery for all legumes. The AS precipitation with 60% to 80% saturation (precipitate IV) resulted in 41-, 88-, and 34-fold of the purity and (−)26%, 126%, and (−)47% of percentage of activity increase for navy beans, red kidney beans, and adzuki beans, respectively. The trypsin inhibitors had a molecular weight of 132 kDa for navy beans, 118 kDa for red kidney beans, and 13 kDa for adzuki beans under nonreducing conditions. The obtained precipitate IV fraction from each legume effectively prevented the degradation of the tilapia muscle with concentration dependent. The myosin heavy chain increased as the concentration of the inhibitor fraction increased, especially at the highest level of addition. The result indicated that the precipitate IV from these legumes have potential for use as a protease inhibitor in fishery related products.     

Enzymatic Reduction of Anti-nutritional Factors in Fermenting Soybeans by Lactobacillus plantarum Isolates from Fermenting Cereals

Soybean is rich in dietary protein but contains some anti-nutritional factors (ANFs), including phytates, tannins, trypsin inhibitors and oligosaccharides. It is used with cereals in weaning foods to improve the protein content and supply essential amino acids. The objective of this work, therefore, was to use microorganisms, specifically Lactobacillus plantarum and the enzymes it produces to reduce anti-nutritional factors and improve the nutritional composition of such food blends. Nine strains of Lactobacillus plantarum isolated from spontaneously fermenting cereals, identified and characterised, were selected based on the abundant production of alpha-galactosidase for the fermentation of the legume. Samples were subjected to fermentation for 5 days and the reduction of anti-nutritional factors was monitored. Anti-nutritional factors and alpha-galactosidase were determined by UV-spectrophotometry. Data were analysed using ANOVA at p = 0.05. Fermentation reduced the tannin content in the raw samples from 1.93 to 0.12 mg/g. Phytate content reduced from 1.16 to 0.04 mg/g. The trypsin inhibitor and protease inhibitor also reduced from 1.20 to 0.010 and 1.2 to 0.020 respectively. The production of alpha-galactosidase by L. plantarum (1.8 unit/ml) enhanced the reduction while the nutritional composition of the food blend in which the soybean was added improved significantly. The use of alpha-galactosidase enzyme by Lactobacillus plantarum from local food sources is thus shown to reduce anti-nutritional factors in soybeans, which can be of benefit in enhancing the nutritional quality of cereal-legume gruels.     

Effect of monoterpenoids on oviposition and mortality of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) under hermetic conditions

Monoterpeniods have been shown to cause mortality in certain stored-product insect pests. The current report investigated the prospects of using monoterpenoids as oviposition deterrents of the cowpea beetle, Callosobruchus maculatus (Fabricius), as well as in the management of populations of the beetle. The monoterpenoids investigated include E-anethole, estragole, S-carvone, linalool, L-fenchone, geraniol, γ-terpinene and DL-camphor, and at the concentrations of 66.7, 33.3, 16.7, 8.33 and 0 μL/L. Exposure of the life stages of the beetle, which included eggs, young larvae (first instar), 4th instar, pupae and adults to different concentrations of the monoterpenoids over 24 h period caused varying levels of mortality. The stages of the beetle that were the least susceptible to the monoterpenoids were the 4th instar, and the pupae, which required high concentrations of the monoterpenoids to achieve 99% mortality. The adults and the eggs exhibited the highest susceptibility to the monoterpenoids. Mated C. maculatus females that were offered cowpea seeds upon treatment with low doses (8.33 μL/L) of the monoterpenoids did not lay eggs, while control female beetles offered untreated seeds laid several eggs. However, mated C. maculatus females laid eggs on cowpea seeds treated with monoterpenoids 3 weeks before to the day of experimentation. The monoterpenoids did not exhibit residual toxicity to the cowpea beetles. These monoterpenoids could be further investigated for the postharvest management of seed beetles of grain legumes.     

Nutritional and anti-nutritional characteristics of mucuna (Mucuna utilis) bean seeds

Some nutritional and anti-nutritional characteristics of mucuna (Mucuna utilis (Wight) Burck) bean seeds were studied. The mature seeds contained 264 g crude protein, 63 g crude fibre, 41 g crude fat, 37 g ash and 595 g carbohydrates kg^?1 DM. The essential amino acid profile compared well with the FAO/WHO scoring pattern except for a deficiency of sulphur-containing amino acids. Mineral composition was similar to those reported for most tropical grain legumes. Raw mucuna seed samples contained moderately high levels of anti-tryptic activity (2170 trypsin units inhibited g^?1 DM), but this was completely destroyed by cooking. The other anti-nutritional factors (phytate, cyanide and tannins) are probably of little nutritional significance provided that the beans are properly processed. The in-vitro protein digestibility of raw and cooked beans were 71·5 and 80·3 %, respectively. In view of the high L-DOPA contents reported in some mucuna cultivars, overconsumption of mucuna beans should be viewed with some caution until suitable processing methods are developed.     

Automatic, rapid screening of seed resistance in cowpea, Vigna unguiculata (L.) Walpers, to the seed beetle Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) using acoustic monitoring

The biomonitor technique was investigated as a rapid and automatic method for measuring the resistance of cowpea varieties to the seed beetle Callosobruchus maculatus. This technique measures the activity of internally feeding insect larvae by counting ultrasonic emissions produced as they feed. Activity throughout the development of C. maculatus larvae in known susceptible and resistant cowpea varieties was recorded. This showed details of the development of each larval instar, and showed clear differences between the resistant and susceptible cowpeas. A rapid method for comparing cowpeas was proposed in which the activity of larvae was recorded for 24 h starting 14 days after oviposition. Using this method, significant differences in activity were apparent between one susceptible and two resistant cowpea varieties. Further comparisons using a randomised block experimental design also showed a clear difference in activity between one susceptible and one known resistant cowpea variety. This experimental protocol took 21 days. It is suggested that the biomonitor offers a promising method for screening seeds for resistance to insect pests with a reduction in the time and effort required over conventional bioassay methods.     

Antioxidant properties of legumes and their morphological fractions as affected by cooking

The antioxidant properties of 6 legumes: green gram (Vigna radiate), red kidney beans (Phaseolus vulgaris), chickpea (Cicer arietinum), red lentil (Lens esculenta), soybean (Glycine max), and moth beans (V. aconitifolia), and their morphological fractions were evaluated before and after cooking. Kidney beans had the highest total phenolic content (8.9 mg ferulic acid equivalents/g), DPPH free radical scavenging activity (38.1%), reducing power (85.5 μmol ascorbic acid equivalents/g), and total flavonoid content (0.9 mg catechin equivalents/g). Highest metal chelating activity was observed for moth beans (93.3%). The antioxidant properties of the seed coat in all the legumes were manifold higher as compared to the whole legume or its respective endosperm and removal of the seed coat significantly reduced the antioxidant activity. Although cooking brought about a reduction in the antioxidant activity of the legumes in majority of the legumes and their fractions, an increase in the metal chelating activity was observed.     

Dietary fibre content of dry and processed beans

Common beans (Phaseolus vulgaris L.) are a good source of protein, vitamins, minerals and especially dietary fibre. As beans are never eaten raw, the effects of soaking, cooking, soaking–cooking and canning on soluble, insoluble and total dietary fibre contents of beans are studied. Total dietary fibre content was determined by enzyme-gravimetric method. The fraction of insoluble dietary fibre was corrected for the content of resistant starch, determined as part of the total starch in insoluble fibre. The results indicate that thermal processing decreased the insoluble fibre content, and consequently the total dietary fibre content of beans. Soaking and cooking of beans significantly (P⩽0.05) increased the resistant starch content. The data on dietary fibre content of processed food are much more relevant than those of raw food. Thus food composition tables should contain as much data on processed food as possible.     

Use of Seed Sprouting in Modification of Food Nutrients and Pasting Profile of Tropical Legume Flours

The study investigates the effect of seed sprouting on minerals, anti nutrients and pasting characteristics of flours produced from some tropical legume seeds. Samples were collected from cowpea (Vigna ungulculata); red kidney beans (Phaseolus vulgaris), and Pigeon pea (Cajanus cajan) and each sample was divided into two portions. A portion was sprouted while the other portion served as the control. The flours from sprouted and non-sprouted seeds were analyzed for the minerals, anti nutritional and pasting properties. The samples were found to differ significantly (p < 0.05) in the minerals, anti nutritional composition and pasting properties of the flours. Sprouting increased the mineral contents of the flours generally the calcium content of non-sprouted red kidney beans (13.8 mg/100 g) increased to 16.1 mg/100 g in the sprouted flour. The haemagglutinin decreased from 40.6 HU/mg in non sprouted red kidney to 6.4 HU/mg in the sprouted. The final viscosity of the non sprouted flours were higher than those of the sprouted, with non sprouted cowpea scoring the highest, 272 RVU, while sprouted red kidney beans had the least, 109.06 RVU, which implies that sprouting caused a reduction in the gel strength and elasticity of the samples. Seed sprouting effectively increased the mineral nutrients and lowered the anti nutrients, but produced steamed paste with poor gel strength.     

不同预处理方法对豆浆中抗营养因子的影响

豆类中除营养成分外也含有多种抗营养因子,制作豆浆时不同预处理方法可能影响到豆浆中抗营养因子的残留量。本文就浸泡、萌发和去皮3种不同预处理方法对豆浆中胰蛋白酶抑制剂、植酸、单宁等抗营养成分的影响进行综述。与干豆制浆相比,浸泡和萌发处理均可降低所制豆浆中抗营养因子的含量,其中萌发大豆处理去除抗营养因子的效果最佳。