Abstract: Background and Objective: Legumes were studied extensively in the past because of their protein contents but they have now become valuable for their low glycemic index (GI), which is attributed to high amylose and resistant starch contents. This study aimed to evaluate the in vitro starch digestibility and estimated glycemic index (eGI) of native starches from five varieties of cowpea (Vigna unguiculata) cultivated in Indonesia. Materials and Methods: Five varieties of cowpea (namely KT4, KT5, KT7, KT8 and KTL) were extracted their starches using wet milling method. The in vitro digestibility and eGI of cowpea starches were determined using method of Englyst and Goni. Results: Cowpea starches had low RDS content, from 4.09% (KT7) to 7.51% (KT4) but high SDS, from 19.66% (KT5) to 27.07% (KTL) and RS, from 65.75% (KTL) to 76.15% (KT5). The RAG and SAG contents of the cowpea starches ranged from 3.77% (KT7) to 6.79% (KT8) and from 18.56% (KT5) to 25.13% (KT8), respectively. The eGI of cowpea starches varied from 45.46±0.23 (KT5) to 48.14±0.38 (KT8). Conclusion: Cowpea starches have high SDS and RS contents and low GI, therefore they are suitable as a dietary carbohydrate alternative for the management of obesity, diabetes mellitus, cardiovascular disease and certain cancers.
INTRODUCTION
Legumes are the second largest daily dietary foodstuff after cereals. Cowpeas (Vigna unguiculata (L.) Walp.) are among the most important food legume crops and are grown in the semi-arid tropics covering Asia, Africa, Southern Europe and Central and South America1. Cowpeas are harvested for their immature pod or mature seeds and are generally consumed after a single or combination of processes, including soaking, boiling, milling, roasting, fermentation, puffing and germinating2,3.
Daily consumption of legumes may restore various physiological states, such as the blood glucose level, blood lipid profile and production of short chain fatty acids in the colon. Therefore, a legume diet has an important role in controlling and preventing various metabolic diseases, such as obesity, diabetes mellitus, coronary heart disease and colon cancer4.
Cowpea seeds contain approximately 53-66% carbohydrate, most of which is found in the form of starch5-7. Starch from legumes, such as cowpea, has high amylose content and C-type starch crystallinity5,8. Previous studies on starches from legumes have reported many unique properties, such as high viscosity, high resistance to swelling and rupture, high gelatinization temperature, fast retrogradation, high elasticity of the gel and highly resistant starch content compared to starch from cereal3,9. These properties are correlated with the high amylose content of the legume starch.
The glycemic index (GI) is a scale to measure the post-prandial glycemic effect after consumption of carbohydrate-rich foods10. The FAO/WHO experts suggested the use of the GI concept for classifying carbohydrate-rich foods to provide a useful means to help people to select the most suitable carbohydrate-containing foods for the maintenance of health and the treatment of several diseases11,12. Prior studies indicated that starches from legume have poorer digestibility than those from cereal; therefore, they can promote slow and moderate post-prandial glucose and insulin responses and have low GI values9,13. The consumption of low GI foods could prevent the emergence of several diseases, such as obesity, diabetes, cardiovascular diseases and even certain cancers14.
Previous studies on cowpea starches, mostly from the African region, focused primarily on their structure and physicochemical properties5-7,15,16. Studies reported that the in vitro starch digestibility and GI of flours and starches from legumes were influenced by their nature, such as their amylose content17,18. Ratnaningsih et al.8 reported differences in the composition, microstructure and physicochemical properties of starches from five cowpea varieties cultivated in Indonesia. In the present study, starches from five cowpea varieties that were reported to have high amylose content were evaluated for their in vitro starch digestibility and estimated glycemic index (eGI) to provide valuable information about the benefits of cowpea-based diets, especially their ability to prevent several degenerative diseases.
MATERIALS AND METHODS
Materials: Cowpea (Vigna unguiculata) varieties (KT4, KT5, KT7 and KT8) were obtained from the Indonesian Research Center for Legumes and Tubers, Malang, East Java, Indonesia and a local variety (KTL) was obtained from a local farmer in Yogyakarta, Indonesia. The five cowpeas varieties were selected based on their highest productivity and nutritional composition. All chemical reagents used in this study were analytical grade.
Starch extraction: Cowpea starches were extracted by wet milling according to Ratnaningsih et al.8. Cowpea seeds were split using a grinder and steeped in distilled water (ratio of seed:distilled water = 1:3) at 4°C for 24 h. The steep water was decanted and the softened pulses were ground using a blender at high speed for 3 min in distilled water (4°C). The slurry was then filtered. The residual pulp was ground using a blender for 3 min in distilled water (4°C) and filtered. The suspensions from the two filter steps were mixed and allowed to settle overnight at 4°C. The supernatant was then drained off. The starch sediment was redissolved in 0.05 M NaOH and kept at 4°C overnight before neutralizing with 2 M HCl to pH 6 at 4°C overnight. The starch sediment was rinsed with distilled water and allowed to settle at 4°C overnight until the settled starch produced a firm and dense deposit on the bottom. The sediment was recovered and dried at 50°C overnight, ground into powder using a blender, sieved through 100 mesh and stored in a sealed container at 4°C until further use.
Determination of in vitro starch digestibility: In vitro starch digestibility was determined using the method of Englyst et al.19 with modification by Chung et al.20. Porcine pancreatic α-amylase (0.45 g, E-PANAA, Megazyme Inc., Ireland) was dispersed in sterile distilled water (4 mL) and centrifuged at 1500×g for 12 min. The supernatant (2.7 mL) was transferred to a beaker glass and amyloglucosidase (0.3 mL, 3260 U mL–1, E-AMGDF, Megazyme Inc., Ireland) and invertase (0.2 mL, 355 U mg–1, E-INVPD2, Megazyme Inc., Ireland) were added to the solution. This enzyme solution was freshly prepared for each determination of starch digestibility.
Starch (100 mg) and 4 mL of 0.5 M sodium acetate buffer (pH 5.2) were added to each test tube. Enzyme solution (1 mL) and 20 glass beads (4 mm diameter) were added to each tube and the tubes were incubated in a shaking water bath (37°C, 120 rpm). After 20 min, aliquots (0.1 mL) were collected and mixed with 1 mL of 80% ethanol. The solution was incubated again in a shaking water bath (37°C, 120 rpm) and an aliquot (1 mL) was collected after 100 min and mixed with 1 mL of 80% ethanol. The shaking of the water bath was not stopped during the sampling period. The aliquot was taken after 20 min and designated as G20 (rapidly available glucose, RAG) and that taken after 120 min was designated as G120 (slowly available glucose, SAG). The G20 and G120 were centrifuged at 1500×g for 2 min to obtain a clear supernatant for glucose determination.
The remaining solution was removed from the shaking water bath, shaken vigorously to break up any large particles and incubated in a boiling water bath for 30 min. The test tubes were shaken again and cooled in ice-water for 15-20 min. Thereafter, 10 mL of 7 M KOH was added to the aliquot, mixed well and incubated in a shaking water bath containing ice-water (5-10°C) for 30 min. An aliquot (1 mL) was collected, added to 10 mL of 0.5 M acetic acid and 0.2 mL of amyloglucidase, incubated at 70°C for 30 min, placed in the boiling water bath for 10 min, cooled to room temperature, diluted with 40 mL of distilled water and centrifuged at 1500×g for 5 min. An additional aliquot (0.1 mL) was then collected for Total Glucose (TG) measurement.
The determination of Free Glucose (FG) was conducted as follows: Starch (400 mg) and 5 mL of 0.5 M sodium acetate buffer (pH 5.2) were added to screw-cap test tubes, shaken well, incubated in a boiling water bath for 30 min and cooled to room temperature. An aliquot (1 mL) was collected and 2 mL of 80% ethanol was added, shaken well and centrifuged at 1500×g for 5 min. The supernatant (1 mL) was taken and 5 mL of distilled water was added and shaken well for the determination of FG. The hydrolyzed glucose content was measured using glucose oxidase-peroxidase reagent (K-GLUC, Megazyme Inc., Ireland). Aliquots (0.1 mL) were collected and 3 mL of GOPOD reagent was added, incubated at 40-50°C for 20 min and cooled at room temperature. Then, the absorbence was measured at 510 nm.
Starch classification based on the rate of hydrolysis included rapidly digestible starch (RDS, digested within 20 min), slowly digestible starch (SDS, digested between 20 and 120 min) and resistant starch (RS, undigested after 120 min). The digestible starch fractions and available starch fractions were calculated as follows:
| (1) |
| (2) |
| (3) |
| (4) |
| (5) |
| (6) |
Determination of the estimated glycemic index (eGI): Determination of the eGI of cowpea starch was conducted using the method of Goni et al.21. Starch (50 mg) and 10 mL of HCl-KCL buffer (pH 1.5) were added to conical tubes and 0.2 mL of pepsin solution (1 g of pepsin (0.7 FIP-U mg–1, EC 3.4.23.1, Merck Inc., Germany) in 10 mL of HCl-KCl buffer; pH 1.5) was added to each sample and incubated at 40°C for 1 h in a shaking water bath. The volume was brought to 25 mL with tris-maleate buffer, pH 6.9. Then, 5 mL of pancreatic α-amylase solution (Sigma A3176, Sigma-Aldrich Inc., US) in tris-maleate buffer containing 2.6 UI was added to each sample and incubated at 37°C in a shaking water bath. Aliquots (0.1 mL) were collected from each sample after every 30 min from 0-180 min and placed in a tube at 100°C and were then refrigerated until the end of the incubation time. Sodium acetate buffer (1 mL, 0.4 M, pH 4.75) was added to each aliquot and 30 μL of amyloglucosidase (Sigma A9913, Sigma-Aldrich Inc., US) was added to hydrolyze the digested starch into glucose after incubating at 60°C for 45 min in a shaking water bath. The hydrolyzed glucose content was measured using the glucose oxidase-peroxidase reagent (K-GLUC, Megazyme Inc., Ireland). The glucose was converted into starch by multiplying to released glucose weight.
The rate of starch digestion was expressed as the percentage of TS hydrolyzed at different times (0, 30, 60, 90, 120 and 180 min). The total starch hydrolysis (%) of cowpea starches at different times were calculated as follows:
| (7) |
The kinetics of in vitro starch digestion were calculated using the non-linear model established by Goni et al.21. The first-order equation is:
| (8) |
where, C is the percentage of starch hydrolyzed at time t (min), C∞ is the equilibrium percentage of starch hydrolyzed after 180 min and k is the kinetic constant. The parameters C∞ and k were estimated for each treatment based on data obtained from the in vitro starch digestion. The area under the hydrolysis curve (AUC) was calculated using the following equation:
| (9) |
where, C∞ is the equilibrium percentage of starch hydrolyzed after 180 min, tf is the final time (180 min), t0 is the initial time (0 min) and k is the kinetic constant.
The hydrolysis index (HI) represents the rate of starch digestion and the predicted GI indicates the digestibility of the cowpea starch in relation to the digestibility of starch in a reference material, white bread. The HI, a good predictor of glycemic response, was calculated by dividing the AUC of each treatment by the AUC of a reference (white bread). The GI was then estimated using the following equation of Goni et al.21:
| (10) |
Statistical analysis: All experiments were performed in duplicate and were expressed as the Mean±Standard Deviation. Data were analyzed using analysis of variance. Duncan's multiple range tests were conducted to assess significant differences among experimental mean values (p<0.05). The Pearson correlation was used to evaluate the correlation between the digestible starch fraction, available glucose fraction and eGI of cowpea starches.
RESULTS AND DISCUSSION
Total starch, digestible starch fractions and available glucose fractions of cowpea starch: The cowpea starch characterization results (TS, RDS, SDS, RS, RAG and SAG) are shown in Table 1. The total starch content ranged from 80.86-85.42%, which was lower than those reported by Huang et al.6 and Adebooye and Singh15. The RDS is starch that is rapidly and completely digested in the small intestine and is associated with rapid elevation of post-prandial plasma glucose22. The RDS content of various Indonesian cowpea varieties varied significantly and ranged from 4.09% (KT7) to 7.51% (KT4). These values were similar to those reported by Sandhu and Lim13 for starches from black gram, chickpea, field pea, lentil, mung bean and pigeon pea but lower than those reported for other legume starches, such as common bean, pinto bean, red kidney bean, black bean and navy bean14,23. Ambigaipalan et al.9, Kaur et al.24 and Hughes et al.25 reported that the starches from chickpea, faba bean, black bean, pinto bean and mung bean contained higher RDS than cowpea starch. The RDS represents the hydrolysis of starch chains at or near the vicinity of the granule surface and was measured chemically during 20 min of enzyme digestion22. Therefore, the period was not sufficient for all of the hydrolytic enzymes to enter the granule interior since diffusion into the substrate must occur prior to the hydrolytic event9. The difference in RDS content among the cowpea starches reflects the interplay between the surface characteristics and the extent of molecular order at the granule surface8.
The SDS content, which is digested more slowly, varied significantly among varieties of Indonesian cowpea starches, with the highest content in KTL (27.07%) and the lowest in KT5 (19.66%). These values were higher than those reported in previous studies for other legume starches14,24 but lower than those reported by Chung et al.20, Ambigaipalan et al.9 and Liu et al.18. The low SDS in cowpea starches reflects the higher crystalline stability and denser packing of double helices within the crystalline domains (both of which restrict the accessibility of amylolytic enzymes towards glycosidic linkages).
| Table 1: | Total starch, digestible starch fractions and available glucose fractions of cowpea starches |
Values are Means±Standard Deviations and different superscripts in the same column are significantly different (p<0.05), TS: Total starch, RDS: Rapidly digestible starch, SDS: Slowly digestible starch, RS: Resistant starch, RAG: Rapid available glucose, SAG: Slowly available glucose |
|
The SDS is generally considered the most desirable form of dietary starch and is beneficial for the management of several diseases; such as obesity, diabetes, cardiovascular diseases and some cancers26.
The RS content of cowpea starches varied significantly and ranged from 65.75% (KTL) to 76.15% (KT5). These values were similar to the values reported in previous studies for other legume starches14,18 but higher than those reported by Kaur et al.27 and Ambigaipalan et al.9. However, the RS content of all cowpea starches was higher than corn starch (24.5%) but lower than potato starch (84.5%)14. Goni et al.28 classified foodstuff based on RS content as follows: Negligible ≤1%, low 1-2.5%, intermediate 2.5-5.0%, high 5.0-15.0% and very high >15%. According to this classification, cowpea starches were categorized as very high RS foodstuff. The RS content of native starches could be influenced by many factors, such as amylose content, crystallinity, crystalline perfection and amylopectin structure5. Ratnaningsih et al.8 reported that the amylose contents of cowpea starches from Indonesia varied from 39.09-42.78% and had CA-type crystallinities. The high RS content of cowpea starches suggests its potential as a functional ingredient for the development of cowpea-based functional food products. The health benefits of RS in the diet are related to its role as a substrate for probiotic microorganisms growth and its effects on hypoglycemia, hypocholesterolemia, increased absorption of minerals and colon cancer prevention5,22,29.
The RAG content of cowpea starches varied significantly from 3.77-6.79%. These values were considerably lower than those of microwave-baked and water-blanched potatoes, which were 20 and 21/100 g, respectively30 and boiled and microwave-cooked taros, which were 11.6 and 15.6/100 g, respectively12. Englyst et al.31 showed that the RAG content is strongly correlated with GI and that it could be a major determinant of the magnitude of GI for most foods that contained carbohydrate. Therefore, food with a lower RAG content has a lower GI and vice versa. The RAG is another index for the evaluation of starch digestibility that reflects the total amount of glucose released into the blood stream from a certain portion of food31. The RAG value includes both the RDS and FG and it was reported to be a good indicator of the blood glucose and insulin response of foods12,31.
The SAG content of cowpea starches varied significantly from 18.56-25.13%. Englyst et al.32 reported that the SAG content of cereal products varied from 0.6-13.8/100 g. Therefore, cowpea starches have a higher content of SAG than cereal products. A high content of SAG corresponds to a low GI, which is rich in slowly released carbohydrates for the maintenance of blood glucose and the insulin response32.
In vitro hydrolysis rate of cowpea starches: In vitro starch hydrolysis rates of cowpea starches are shown in Fig. 1. The hydrolysis of cowpea starches and white bread increased with digestion time. Cowpea starches showed a lower starch hydrolysis rate than white bread at all times.
| Fig. 1: | In vitro starch digestibility of cowpea starches from different cowpea varieties. Values are the mean of two replicates |
The starch hydrolysis rate of cowpea starches was similar to that of raw mung bean starch24 but lower than faba bean, black bean, pinto bean starches9 and pea starches18. Hoover et al.5 reported that native legume starches were more digestible than potato or high amylose corn starch but less digestible than cereal starches. The lower digestibility of native legume starches has been attributed to the absence of pores on the granule surface, the high content of amylose, B-type crystallites and strong interactions between amylose chains5. The high content of amylose and RS, CA-type crystallinity, smooth surface of the granules and mean granule diameter (Z average, 7.91-15.51 μm) of cowpea starches have been associated with reduced susceptibility to enzymatic hydrolysis8. Differences in the digestibility of native starches among species have been attributed to the interplay of multiple factors, such as the starch source, granule surface organization (e.g., pores), granule size and architecture, amylose/amylopectin ratio, retrogradation of amylose, amylose-lipid complexes, amylose chain length, molecular structures of amylopectin, degree of crystallinity, type of crystalline polymorphic forms (A, B or C), the amount of B-type crystallites in C-type starches and the presence of compound granules5,18.
Estimated glycemic index (eGI) of cowpea starches: The kinetic constant, HI and eGI of cowpea starches are shown in Table 2. The kinetic constant of the starch hydrolysis of cowpea starches varied from 0.0261±0.0020 to 0.0328±0.0002, which was higher than those reported by Goni et al.21. The HI represents the digestibility of starch in foods in relation to the digestibility of starch in a reference food, namely, white bread13. The HI of cowpea starches varied significantly from 10.47±0.42% (KT5) to 15.36±0.69% (KT8). These values were in the range reported by Kaur et al.27 for lentil starch and by Kaur et al.24 for mung bean starch but much lower than bean starch23 and oat starch33.
The eGI of the cowpea starches varied significantly from 45.46±0.23 (KT5) to 48.14±0.38 (KT8). These values were similar to those reported by Sandhu and Lim13 and Kaur et al.27,24 but much lower than those reported by Chung et al.23. Foster-Powell et al.11 classified the glycemic index of foods as follows: Low (GI<55), medium (GI 56-69) and high (GI>70). According to this classification, all of the cowpea starches were categorized as low GI. The low GI of cowpea starches has been attributed to the high content of amylose and RS, CA-type crystallinity, the smooth surface of the starch granules and the strong interactions between amylose chains. The reported health benefits of low-GI starchy foods include improved blood glucose control, reduced insulin demand, reduced blood lipid levels in healthy adults and patients with diabetes and hypertriglyceridaemia, improved satiety and increased colonic fermentation34.
| Table 2: | Kinetic constant (k), calculated hydrolysis index (HI) and estimated glycemic index (eGI) of cowpea starches |
Values are Means±Standard Deviations and different superscripts in the same column are significantly different (p<0.05) |
|
| Table 3: | Pearson correlation among starch digestible fraction, available glucose fraction and estimated glycemic index of cowpea starches |
*,**Significant correlation at p<0.05 and p<0.01, respectively, SDS: Slowly digestible starch, RS: Resistant starch, RAG: Rapid available glucose, SAG: Slowly available glucose, eGI: Estimated glycemic index |
|
Therefore, consumption of low-GI foods could play an important role in the management and prevention of several degenerative diseases, such as obesity and diabetes.
Correlation between the starch digestible fraction, available glucose fraction and estimated glycemic index: The correlations among the starch digestible fraction, available glucose fraction and eGI of cowpea starches are shown in Table 3. The RDS and SDS were negatively correlated with RS (r = -0.689 (p<0.05) and r = -0.914 (p<0.01), respectively). The RDS was positively correlated with RAG (r = 0.996, p<0.01), where RAG increased as RDS increased. SDS was positively correlated with SAG and eGI (r = 0.984 and 0.797 (p<0.01), respectively). The RS was negatively correlated with RAG, SAG and eGI (r = -0.683 (p<0.05), r = -0.895 and -0.819 (p<0.01), respectively). A higher RS content in cowpea starches was associated with lower RAG, SAG and eGI. The SAG was positively correlated with eGI (r = 0.768, p<0.01). These results were in agreement with previous studies of Sandhu and Lim13 and Nayak et al.34.
CONCLUSION
The in vitro starch digestibility and eGI varied widely among the five species of Indonesian cowpea. All cowpea starches had low RDS, RAG, HI and eGI and high SDS, RS and SAG. The variety KT5 had the highest RS and the lowest eGI, which are the ideal characteristics. Correlation analysis of cowpeas starch digestibility and its GI indicated a strong negative relationship between the RS content and RAG, SAG and eGI. This study suggested that because of its low GI and other characteristics, cowpea starch is strongly recommended for use in functional food formulations for the prevention of several diseases, such as obesity, diabetes, cardiovascular diseases and some types of cancer.
SIGNIFICANCE STATEMENTS
| • | In vitro starch digestibility and estimated glycemic index (eGI) of cowpea starches were evaluated |
| • | Cowpea starch had low Rapidly Digestible Starch (RDS) and Rapidly Available Glucose (RAG) contents but high Slowly Digestible Starch (SDS), resistant starch and Slowly Available Glucose (SAG) contents, thus, it had low GI |
| • | The low GI of cowpea starch is strongly recommended as a functional ingredient to develop a new functional food for the prevention of several degenerative diseases, such as obesity and diabetes |
ACKNOWLEDGMENT
The authors gratefully acknowledge to the Directorate General of Higher Education, Ministry of Research, Technology and Higher Education, Republic of Indonesia, for supporting the Doctoral Fellowship under which the present project was carried out.