Abstract: This study describes the isolation, digestibility and effect of process conditions on the Parkia biglobosa (African locust bean) starch digestibility. Parkia starch fractions are: Total Starch (TS), Rapidly Digestible Starch (RDS), Slowly Digestible Starch (SDS) and Resistant Starch (RS). The results indicate that processing conditions can be changed to effectively control the relative content of SDS and RS in Parkia starch products. Amylose is the molecular basis of RS while amylopectin is the main constituent of SDS and plays a key role in the structure and digestibility of SDS. This methodology may enable process modifications to influence the functional digestibility properties of prepared Parkia starch products.
INTRODUCTION
Africa locust bean (Parkia biglobosa) is a perennial tree legume which belongs to the sub-family Mimosoideae and family Leguminosae. It grows in the savannah region of West Africa (Pelig-Ba, 2009; Ihegwuagu et al., 2009). Africa locust bean tree is an important food tree for both man and livestock such as husks and pods and plays a very vital role in the rural, corresponding author economics of West African countries; virtually every part of the species is of value as food or fodder (Teklehaimanot, 2004; Tee et al., 2009). Starch is the major storage carbohydrate in plants. It is produced as granules in most plants cells and is referred to as native in this state. Native starches from different botanical sources vary widely in structure and composition but all granules consist of two major molecular components, amylose (20-30%) and amylopectin (70-80%) (Sankhon et al., 2012). The physicochemical properties of starch and its use depend largely on its biological origin and source and the various sources include cereal, grain, nuts, seeds, leaves, tubers and root. Because starch finds application in various industries, the researches for new sources of starch, like P. biglobosa, becomes necessary. Although qualitative determination of the chemical and nutritional composition of P. biglobosa seeds revealed that it is rich in starch, lipids, protein, carbohydrates, soluble sugars and ascorbic acid (Pelig-Ba, 2009; Ihegwuagu et al., 2009). Studies indicate that the digestibility of starch-based products are not only affected by food type, degree of maturation, starch structure, starch content, food ingredients and individual factors (FAO, 1998), processing and storage, pea starch is very susceptible to retrogradation and thus becomes rich in RS which is naturally indigestible (Skrabanja et al., 1999). The retrogradation rate and extent are mainly affected by the inherent starch properties, including molecular and crystalline structure and by storage conditions, such as temperature, time and water content (Gudmundsson, 1994; Liu and Thompson, 1998). Amylopectin contributes to the retrogradation occurring in long-term rheological and structural changes, whereas amylose is usually responsible for the short-term, rapid changes in food texture (Sathaporn and Jane, 2007). Starch often changes from an amorphous state to a crystalline state and thus this retrogradation process includes a recrystallization (Yuan et al., 1993).
In this research, the Parkia starch powder was prepared to apply in the food industry via. a soaking and cooking process which differed from normal domestic preparation, therefore, the objectives of this study are to isolate and investigate how different process conditions affect on the digestibility of Parkia biglobosa starch and to reduce slow starch digestibility in favor of resistant starch.
MATERIALS AND METHODS
Africa locust bean (Parkia biglobosa) seeds were purchased from the local market in Madinah (Conakry, Guinea) in August, 2011 and shipped to Wuxi, China. Porcine (invertase, pancreatic α-amylase, amyloglucosidase) were purchased from Sigma Aldrich Co. Ltd. (Shanghai, China). The other chemicals, potassium hydroxide, sodium hypochlorite, ethanol, 3,5-dinitro salicylic acid were purchased from Sinopharm Chemical Reagent Co. Ltd. All other reagents used were of analytical grade.
The different samples ratio material/water (1:2, 1:3, 1:4, 1:5, 1:6 and 1:7 w/v) were prepared in a sterilization equipment chamber pot (YXQ-LS-SII shanghai Boxun, industry and trade Co. Ltd., medical equipment factory). The following conditions were applied: time of soaking (10 h) and cooking temperature (90°C) for 3 h. The samples were cooled at room temperature (28°C) and then placed in a refrigerator (4°C) for 72 h. Samples were dried in a hot air oven at 45°C until constant weight, after cooling in a desiccator the samples were ground and sieved using 60 mesh sieve and stored in sealed plastic bags until analysis for starch digestibility. The different samples ratio were taken one by one for the fraction of starch digestibility (RDS, SDS and RS). All samples were treated in triplicate.
This research was conducted in the School of Food Science and Technology Laboratory and State Key Laboratory of Jiangnan University, Wuxi from August 2011 to May 2012.
Starch isolation: The isolation of starch from Parkia biglobosa seed was performed according to the method of Sira and Amaiz (2004) with slight modification. Visible dirt and contaminants were removed from the dark-colored Parkia seed (1 kg) which was then steeped in a solution of sodium hypochlorite (35 g) and potassium hydroxide (50 g) in water (2 L) at room temperature (28°C) for 3 h. The pH of the steep solution was elevated to 9 and the mixture was maintained at 100°C in a thermostat water bath for 3 h. Then, the solution was drained and the seeds were immersed in water and left overnight at ambient temperature. Finally, the seeds were thoroughly washed, manually dehulled and the cotyledon was washed repeatedly until the wash pH was neutral. The cotyledon was blended with water for 24 h using a domestic blender. The homogenate was filtered through muslin cloth and the filtrate was allowed to settle overnight. The supernatant was decanted and the sediment was centrifuged at 4500 rpm for 10 min using a ZOPR-52D refrigerated centrifuge (Hitachi Koki Co. Ltd., Tokyo, Japan). The sedimented starch was re-suspended in water and the process was repeated six times. The resultant starch was dried at 60°C in a hot air oven, then grounded to powder using a mortar and pestle and stored in cellophane and wrapped before usage.
In vitro digestibility of Parkia starch: Five hundred milligrams of starch fractions: Total Starch (TS), Rapidly Digestible Starch (RDS), Slowly Digestible Starch (SDS) and Resistant Starch (RS) were measured. Samples were incubated with invertase, pancreatic α-amylase and amyloglucosidase at 37°C in capped tubes immersed in a water bath shaker according to the method of Englyst et al. (1992) and the supernatants were measured at 0, 20 and 120 min for glucose content. The glucose data obtained was used to calculate the content of various starch types using the following formulas:
Total starch (TS) = (Total glucose (TG)-free glucose
(FG))x0.9 Rapid digestible starch (RDS) = (G20-FG)x0.9 Slow digestible starch (SDS) = (G120-G20)x0.9 Resistant starch (RS) = TS-(RDS+SDS) |
where, FG, G20 and G120 (mg) represent the amount of glucose in the supernatant at 0, 20 and 120 min of hydrolysis, respectively. Total Glucose (TG) was measured with the 3,5-dinitrosalicylic acid according to method of Rose et al. (1991) after the starch was completely hydrolyzed into glucose by perchloric acid. The in vitro digestibility of starch was determined from the calibration curve equation:
y = 0.743x+0.0004 and R2 =0.9958 |
Processing conditions of starch digestion: To improve the yield of modified starch from Parkia seeds, different parameters were taken in consideration according to the digestion method of Englyst et al. (1992). The sample/water ratio were fixed as (1:2, 1:3, 1:4, 1:5, 1:6 and 1:7) w/v, the soaking time (6, 8, 10, 12 and 24 h) and the cooking temperature (50,70, 90, 110 and 121°C) for 3 h. Then, the optimal operating conditions was determined by means of varying one parameter while keeping the others at a constant level. First the sample/water ratios were varied, after finding the best ratio; followed by cooking temperatures for 3 h and finally soaking times were varied to study the effects that affect on the digestibility of Parkia biglobosa starch.
Differential scanning calorimetry (DSC): Calorimetric measurements (gelatinization temperature and enthalpy) of the processed Parkia starch product with conditions of material/water ratio 1:4, soaking for 12 h, 110°C cooking for 3 h and the samples were cooled at room temperature (28°C) and then placed in a refrigerator (4°C) for 72 h were analyzed with the Pyris-1 Differential Scanning Calorimeter (DSC) (PE, USA). Samples (3 mg) were weighed directly into DSC aluminum pans and deionizer water, after sealing, the pans were left to equilibrate (24 h) at room temperature and then heated from 30 to 120°C at 10°C/min. In all measurements an empty pan was used as reference. Onset (To), peak (Tp) and Endset temperature (Te) of gelatinization as well as gelatinization enthalpy changes (DH) were determined.
Statistics: The test results were processed by using One-way Analysis of Variance (ANOVA) test using statistical software (SAS, version 8.1). Differences at p<0.05 were considered to be significant.
RESULTS AND DISCUSSION
In vitro digestibility of Parkia starch: Based on the Englyst test, percentages of RDS, SDS and RS in normal Parkia starch were 10.91, 52.29 and 33.41%, respectively exhibited significant difference (p<0.05).
Our results corroborate with the work of Zhang et al. (2006, 2008) in waxy maize, wheat, rice and maize. The RDS level (10.91%) in the Parkia starches were generally lower than those reported for pea (18.2-23.8%), lentil (16.0-16.9%) and cultivars of other chickpea (21.5-29.9%) starches (Chung et al., 2008a). The SDS (52.29%) was comparable to those of pea (53.7-59.0%) lentil (58.3-62.2%) and other chickpea cultivars (45.7-57.7%). Whereas, RS (33.41%) were much higher than those reported by Chung et al. (2008a, b) for pea (8.1-12.6%), lentil (13.0-13.2%) and other chickpea cultivars (8.4-18.4%).
SDS content which is considered a desirable form of dietary starch, was 52.29%. This value are much higher than those reported by Zhang et al. (2006) for maize (53.0%), waxy maize (47.6%), wheat (50.0%), rice (43.8%) and potato (15.2%). RS content which is considered a desirable form of dietary starch, was 33.41%. This value is much higher than those reported by Pongjanta et al. (2009) of bean varieties AC Nautica (17.2%), Majesty (21.3%) and red Kanner (21.9%); although measured using the Englyst method. The RDS, SDS and RS (Fig. 1) levels of the Parkia starches cannot be compared with those reported for other legume starches, due to differences in methodology AACC (2000) vs. Englyst et al. (1992) and to different time periods of hydrolysis that have been defined for measurement of RDS, SDS and RS levels.
Generally, digestibility of native starch is influenced by starch source, granule size, amylose/amylopectin ratio, crystallinity and amylopectin molecular (Chung et al., 2006; Singh and Ali, 2006). SDS which leads to a slower entry of glucose into the blood stream and a lower glycemic response, is digested completely in the small intestine at a lower rate as compared to RDS, while RS is the starch portion that cannot be digested in the small intestine but is fermented in the large intestine (Oates, 1997; Amadou et al., 2011).
A moderate postprandial glycemic and insulinemic response of SDS implies that SDS rich foods may provide wide health benefits in reducing common chronic diseases such as obesity, diabetes and cardiovascular disease through lessening the stress on regulatory systems related to glucose homeostasis (Ludwig, 2002).
| Fig. 1: | Digestibility of Parkia starch by Englyst test |
| Table 1: | Effect of the sample cooking (material/water ratio) on in
vitro digestibility of Parkia starch soaked at 10 h, cooked at
90°C for 3 h and after storage at 4°C for 72 h |
| Values (Mean±SD) in the same column with different letters are significantly different at p<0.05, n = 3 | |
Effect of sample/water ratio on the starch digestion: The results exhibited that ratio 1:4 (Table 1) as the best ratio for the digestion of Parkia starch. There are significant differences (p<0.05) among the values 12.09, 39.76 and 44.76%, respectively for RDS, SDS and RS. These results are comparable to those reported by Yao et al. (2010) and Zhang et al. (2008). Various studies have shown that several factors can contribute towards the differences in RS quantities in foods such as: botanical origin; nature of starch (amylose and amylopectin content and their ratio); food processing (degree of starch gelatinization and retrogradation); starch morphology (particle size and cellular structure), types of starch granules or their crystalline structure (such as A, B and C or V) and presence of other components (lipids, protein, dietary fiber, antinutrients and organic acids etc.) (Goni et al., 1997). The material/water ratio might affect the space available for starch chain extension in the water/starch suspension. At high concentrations, starch chains may not be fully extended; and at low concentrations, extended linear and branched starch molecules have less probability to contact each other thus limiting the formation of organized molecular arrangements and crystals and subsequently forming high levels of RS (Yao et al., 2010).
Previous research have demonstrated that in processed food RDS and glycemic index are highly correlated; while, SDS from the structural perspective show and prolonged release of glucose of native maize starch. Furthermore, RS is resistant to digestion and no glucose is available for glycemic response. However, the fermentation of RS in the large intestine generates short-chain fatty acids that are beneficial for colonic health. Both SDS and RS either alone or in combination, contribute to an improved nutritional quality of starch (Englyst et al., 1999; Seal et al., 2003; Zhang et al., 2006; Topping and Clifton, 2001).
Effect of cooking temperature on starch digestion: After choosing the ratio (1:4), the temperatures were varied (50, 70, 90, 110 and 121°C) for 3 h and the soaking time was kept for 10 h and continued the process as in previous first case (material/water). The result showed in (Fig. 2) that the ratio (1:4), the temperature of 110°C was the best with RDS, SDS and RS, 12.97, 37.99 and 45.65%, respectively which are significantly different (p<0.05). The proportions of RS of cooked temperature (90, 110 and 121°C) starch samples are good results, exhibiting the best result of 45.65% digestibility. However, when compared to results following the proportion of SDS and RS fractions of pea starch (8.5 and 47.7%, respectively) (Yao et al., 2010) which are slightly lower than our data in the same proportion of SDS and RS starches used.
| Fig. 2: | Effect of the cooking temperature on the in vitro digestibility
of Parkia starch, with material/water ratio; 1:4, soaking time; 10
h and cooking temperature; 50, 70, 90, 110 and 121°C for 3 h, after
storage at 4°C for 72 h |
Pongjanta et al. (2009) commented on the proportion of RDS, SDS and RS fractions in legume starches that it is difficult to make a meaningful comparison of the levels of RDS, SDS and RS proportion of legume starch since most of these studies used different digestibility methods varying in time of hydrolysis and enzyme sources. High temperature and high pressure treatment would lead to full gelatinization of starch granules and complete migration of amylose molecules. The more, cooking temperature the lower the viscosity which lead to more free starch. And when lower viscosity, free starch (such as amylose) molecules have easier access to each other and tend to form inter-molecular hydrogen bonds which is favorable to full association of amylose double helix molecules and the formation of RS. Too high a process temperature will give further decrease of starch molecules to provide low molecular weight hydrolyzates with limited potential for re-associations (Chinma and Igyor, 2008; Oates, 1997; Goni et al., 1997). Chung et al. (2009) stated that the amylose-amylose interactions which are much stronger than those of amylose-amylopectin or amylopectin-amylopectin, may have continued to exist after gelatinization and thereby partly restricting accessibility of starch chains to the hydrolyzing enzymes.
Effect of soaking time on the digestion of Parkia starch: The results of RDS, SDS and RS for soaking time were 12.56, 37.82 and 46.22%, respectively. The best soaking time for the digestion of Parkia starch appear to be 12 h with significant (p<0.05) increase in starch digestibility as it is shown in Fig. 3. Similar phenomenon was observed in the work of Siddhuraju and Becker (2001) where soaked and boiled legumes had significant increase in digestibility.
Soaking affect the starch digestibility, involves entry of water into the legume kernels, wetting and dissolving soluble nutrients. The observed changes in RS and SDS level may be explained by the relatively slow cooling after gelatinization, starch molecules became less energy active and starch molecules of appropriate sizes rearranged to form orderly crystalline precipitation which made starch paste retrograde into a gel. Although starch is not generally soluble in cold water, our study showed that soaking significantly (in most cases) increased all starch fractions in all sample (Eyaru et al., 2009). The soaking of seeds in plain or tempered water produces a swelling of the tissues and water uptake without cell separation; Bishnoi and Khetarpaul (1993) found that digestibility increased with soaking of all peas studied.
| Fig. 3: | Effect of the soaking time on the in vitro digestibility
of Parkia starch with material/water ratio; 1:4, soaking time; 6,
8, 10, 12 and 24 h and cooking temperature; 90°C for 3 h, after storage
at 4°C for 72 h |
The observed changes in RS and SDS level may be caused by the relatively slow cooling after gelatinization, starch molecules became less energy active and starch molecules of appropriate sizes rearranged to form orderly crystalline precipitation which made starch paste retrograde into a gel; as soaking time prolonged, the bundle structure formed between starch chains by hydrogen bonding may dissociate which is not conducive for the formation of RS but rather favors the formation of SDS. Conversely, the results of Eyaru et al. (2009) contributed to the different consequence of soaking and cooking. They soaked Parkia starch before cooking and this resulted in reduced starch fractions, possibly due to leaching of soluble fractions.
Differential scanning calorimetry (DSC): DSC is usually a tool used to investigate the phase transitions of the gelatinization process and it has been reported earlier that Parkia starch is a less stable one (Ihegwuagu et al., 2009). The thermal properties of Parkia starches; onset, peak and endset temperatures (To, Tp and Te, ΔH, respectively) are summarized in Table 2. This corroborates our earlier observation that to heat than corn starch (Ihegwuagu et al., 2009).
In principle, fully gelatinized starch should produce a flat straight line with no absorption peak in DSC analysis. However, starch molecules rearrange and retrograde to form many crystal-like structures; breaking these crystal structures to re-solubilize starch molecules requires external energy. The gelatinization transition temperatures [onset temperature (To), peak temperature (Tp), Endset temperature (Te) and the enthalpy of gelatinization (ΔH) of native and modified starches are presented in Table 2. Cooking starch influenced the gelatinization properties of all the samples. The gelatinization temperature of native Parkia starch ranged from 83.65-86.23°C which corroborate with those of other tuber starches, such as potato, cassava and new cocoyam (Collado et al., 1999; Oladebeye et al., 2009; Shi and Seib, 1992). The onset temperature for melting retrograded amylopectin is in the range of 63.74 to 66.30°C. In addition, Table 2 shows that after Parkia processing, its (ΔH) increased in parallel with its content of RS and SDS which led to decrease in digestibility of the final Parkia starch product, thus, this may be attributed to retrograded amylopectin.
| Table 2: | Differential scanning calorimetry (DSC) of Parkia
native starch and cooked starch |
| Values (Mean±SD) in the same column with different letters are significantly different at p<0.05, n = 3 | |
CONCLUSION
In conclusion, the study showed that the ratio 1:4; soaking time 12 h at 110°C for 3 h as the best conditions to produce the high Parkia starch digestion. However, cooking and processing can be carried out in various ways and under a range of condition, so that the products can vary widely in the degree of denaturation, gelatinization and retrogradation they have undergone.
Modifications in processing conditions for Parkia biglobosa starch product exhibited minimal impact on the content of RDS though had specific effect on the SDS and RS content. Results deduced that amylose is the molecular basis of RS and amylopectin plays a key role in the structure of SDS and is the main constituent of SDS. SDS and RS significantly indicated that various processing conditions promote the inter-conversion between them. Thus, processing conditions can be changed to effectively control the relative content of SDS and RS in Parkia starch products. This methodology may enable process modifications to influence the functional digestibility properties of prepared Parkia starch products.
ACKNOWLEDGMENTS
This article is supported by 111 project-B07029 and PCSIRT062, China. The authors are also grateful to Fatoumata Sylla manager at ministry of Foreign Affairs Conakry, Guinea who sent the sample down to Wuxi, China.