Impact of Process Conditions on Digestibility of Pea Starch

Abstract

This study investigated how process conditions affect the digestibility of pea starch from pea starch powder (PSP). The factors considered were resistant starch (RS), slow digestible starch (SDS) and rapidly digestible starch (RDS) content. The examined five process factors were: material/water ratio, cooking temperature, cooking time, soaking time, and heat dehydration time. Changes in process conditions mainly altered the content of RS and SDS. Analysis with Sephadex G-200 chromatography and differential scanning calorimetry revealed that RS was mainly from retrograded amylose and amylopectin, while SDS and RDS were mainly derived from amylopectin.

Keywords:

• Pea
• Resistant starch
• Slow digestible starch
• Amylose
• Amylopectin

INTRODUCTION

Pea (Pisum Sativum L.) is an edible legume and it has multiple uses including human food, animal feed. Its yield, as total world crop, ranks the fourth in the legume family, after soybean, peanut, and string bean.[1] Pea is a cool-season legume crop produced throughout the world in cool temperate climates. Normally, human consumption of pea product is as a domestic food material, and there are no abundant industrial products. The consumption of pea is not so optimistic, for example, pea production in 2007 at max. 45.000 MT decreased down from 80.000 MT in 2001 in Czech Republic.[2] So, there is a demand to develop the pea resource.

Pea seeds are high in starch content, usually reaching 55% ∼ 70% and are low in glycemic index as compared with the most commonly consumed grains.[1] The blood glucose response of the human body to foods largely depends on the rate of digestion. Studies indicate that the results of in vitro digestion experiments are clearly correlated with glycemic index,[3] and foods high in resistant starch (RS) and slow digestible starch (SDS) possess low glycemic indices. It has been demonstrated that eating low glycemic index foods offers some therapeutic effects for non-insulin-dependent diabetes and cardiovascular diseases.

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,[4] but also by how foods are processed.[5] It has been reported that heat-moisture treatment increases the content of RS in peas;[6,7] thermal processing (normal cooking) significantly increases the content of rapidly digestible starch (RDS) and decreases RS fractions in native pea starch[8]; and during processing and storage, pea starch is very susceptible to retrogradation and thus becomes rich in RS, which is naturally indigestible.[9]

In this research, the pea starch powder (PSP) was prepared to apply in the food industry via a cooking, soaking and heat dehydration process, which differed from normal domestic preparation. This study focused its investigation on how different process conditions affect the in vitro simulated digestibility of starch in the PSP product, and offers discussion to explain the mechanism at a molecular level.

MATERIALS AND METHODS

Materials

Dried wrinkled peas (Dried under the sun to moisture content 10% after harvest, packaged in plastic bag and stored at room temperature) grown in Jiangsu, China were used. Porcine pancreatic α-amylase and glucoamylase were from Sigma (US). The other chemicals, 3,5-dinitro salicylic acid, glycerol, sodium hydroxide, methanol, anhydrous ethanol, lead acetate, sodium sulfate, were of chemical pure or analytic grade and were purchased from Sinopharm Chemical Reagent Co., Ltd.

Preparation of Pea Starch Powder (PSP) and Single-factor Experiments

The flow chart for preparation of the PSP product is shown in Fig. 1. The dry peas (50 g) were cooked in water contained in a steam sterilization pot (YXQ-LS-50SII, Boxun Industry & Commerce Co., Ltd., Shanghai) until they had completely softened. Cook water was discarded, and the solids were put into a spin filter where the coat and embryo (hypocotyl) were separated from the remaining slush. The centrifuged slush was further soaked for a specified period, spun to remove most of the water, put in an electric frying pan (12-inch inner diameter, Kema company, Guangdong), and heated continuously; it was stirred until it was dry. The heating surface temperature was about 160°C, and the material was stirred at 30 r/min with a blender for 30 min. The dehydrated pea solids were naturally cooled, milled, collected, and passed through a 40-mesh sieve, which yielded the final pea starch powder (PSP) product for analysis.

Figure 1 The flow chart of preparing the PSP product.

Based on our preliminary work of preparing PSP, one process factor was examined each time while other factors were held constant. The parameter ranges of the factors were: material/water ratio, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8; cooking temperature, 101, 108, 111, 118, and 121°C; cooking time, 2, 2.5, 3, 3.5 h; soaking time, 1, 3, 5, 7, 24 h; heat dehydration time, normally stir-heat to dryness (for 50 g dry peas, it usually requires approximately 30 min), and then continue stir-heat till 45 min. Each experiment was replicated twice.

Determination of in vitro Simulated Digestibility

Englyst method[3] was used for simulating digestibility. The PSP products obtained from the differential process conditions were separately digested at pH 5.2 and 37°C by an enzyme cocktail (pancreatic α-amylase, glucoamylase, invertase) and the supernatants were measured at 0 min, 20 min, and 120 min for glucose content. The glucose data obtained was used to calculate the content of various starch types using the following formulas:

(1)

(2)

(3)

(4)

In the formulas, FG, G₂₀ and G₁₂₀ were the amount of glucose in the supernatant at 0 min, 20 min and 120 min of zymolysis (mg), respectively. Total glucose (TG) was measured by the 3,5-dinitrosalicylic acid method after the starch were completely hydrolyzed into glucose by perchloric acid.[10] The linear equation for the determination was y = 0.59353x + 0.0124, and R² = 0.9987. Each experiment was replicated twice.

Sephadex G-200 Classification of Starches of Different Digestibility

The samples were prepared under the process conditions for optimum RS formation （(50 g dry pea cooked at 121°C for 3 h with material/water ratio of 1:6, soaked for 5 h, normal heat dehydrated for 30 min)） and characterized following 0 min, 20 min, and 120 min enzymatic digestion. They were centrifuged, the supernatants were discarded, and the pellets were washed and centrifuged again to yield the unhydrolyzed substrates. The substrates left after 20 min zymolysis were defined as SDS plus RS and the substrates left after 120 min zymolysis were defined as RS. The 0 min pea material was used as the control. They were separately dissolved in dimethylsulfoxide (DMSO).

The samples from three digestion times were independently loaded (1 mL) onto a Sephadex G-200 column (1.5 cm × 100 cm), eluted with 50 mmol/L NaCl (containing 0.02% sodium azide) at a rate of 0.4 mL/min, and each collection tube contained 4 mL. Sugar content was determined by the phenol-sulfuric acid method. A standard curve was generated with glucose. The collection tubes containing a peak were pooled, and the pooled samples were reacted with iodine reagent (mixtures of 2 mg/mL KI and 0.2 mg/mL I₂). A blue color reaction indicated amylose while a purple reaction indicated amylopectin.[11]

Analysis of Thermodynamic Properties

Thermodynamic properties of two pea samples, the raw material (wrinkled dry peas) and the processed PSP product (with material/water ratio 1:6, 121°C cooking for 3 h, soaking for 5 h, and normal heat dehydration for 30 min to dryness), were analyzed with the Pyris-1 differential scanning calorimeter (DSC) (PE, USA). The instrument was first calibrated with metal indium, then a sample was precisely weighted and added into the aluminum crucible, and a defined amount of pure water (sample: water ratio 1:4 (w/w)) was added and they were mixed to homogeneity. The crucible was capped and tightly sealed, and was allowed to equilibrate for 24 h at room temperature. An empty crucible served as the control. The crucibles were heated from 40°C to 100°C at a rate of 10°C/min. Each sample was measured in triplicate and the mean and standard deviation (SD) were reported using the software SPSS 16.0.

Statistical Analysis

The test results were processed by using one-way variance analysis (ANOVA). Differences at p < 0.05 were considered to be significant. The employed software was SPSS 16.0.

RESULTS AND DISCUSSION

Source of RS in Processed PSP Products

Zymolyzed PSP samples classified by Sephadex G-200 demonstrated that solutions corresponding to peak 1 were amylose due to their ability to form the characteristic complex with iodine. In contrast, solutions of other peaks (peaks 2–6) did not have this property and therefore were amylopectin (Fig. 2). Figure 2 displays the peak profile of processed PSP samples after 0, 20, and 120 min of zymolysis. Generally, amylopectin has large molecular weight (MW) and should be eluted first; amylose has small MW and should be in the last elution peak; and the partially hydrolyzed components have MW between amylopectin and amylose and therefore their elution peaks should be between the two. [12,13]

Figure 2 Sephadex G-200 chromatogram (490 nm) of starch of PSP product before zymolysis (0 min) (A), at 20 min (B) and 120 min (C) after zymolysis.

As shown in Fig. 2A, processed PSP products mainly contained two MW components, which were amylopectin (peak 1) and amylose (peak 6). After 20min of zymolysis, by definition, RDS were enzymatically hydrolyzed and the remaining portion was designated as SDS and RS. The corresponding chromatogram (Fig. 2B) showed a smaller amylopectin peak, a small new peak appearing at tube 15 and 16 (peak 2), and an unchanged amylose peak (peak 6). This suggested that within 20 min of zymolysis the reduced sugar measured in the supernatant (defined as RSD) was provided by amylopectin and the RSD portion was zymolyzed into glucose and amylopectin of smaller molecular weight (peak 2). After 120 min of zymolysis, by definition, both RDS and SDS were zymolyzed and the remaining portion was RS. The corresponding chromatogram (Fig. 2C) showed a much smaller amylopectin peak (peak 1), three even smaller peaks (peaks 3, 4, and 5) of MW between peaks 2 and 6, and an unchanged amylose peak (peak 6). This suggested that the reduced sugar measured in the supernatant corresponding to SDS was also provided by amylopectin. Therefore, it could be said that RDS and SDS in processed PSP products were both derived from amylopectin while the source of RS was primarily amylose and partially amylopectin.

Relationship between Thermodynamic Properties and Digestibility of Peas before and After Processing

The thermodynamic properties of pea before and after processing were analyzed with DSC. In DSC analysis, higher gelatinization temperature (T) indicates higher degree of intactness of the starch crystal, and higher phase transition enthalpy (δH) indicates higher content of starch crystal. The DSC analysis results presented in Table 1 showed that the DSC spectrum of raw pea seed contained an obvious heat-absorption peak (Tp, 77.0°C), in contrast to the spectrum of processed PSP product, which did not contain such an obvious peak (Tp, 55.4°C). Chung et al.[14] also reported that DSC heating thermogram showed a single endothermic peak (60–80°C) for native pea starch, and this starch gelatinization temperature differed for cultivars within the same species.[15]

Table 1 Thermodynamic properties of pea sample before and after processing

Sample	Δh (J/g)	To (°C)	Tp (°C)	Tc (°C)	RS (%)	SDS (%)
Raw pea sample	3.0 ± 0.2^a	75.8 ± 0.4^b	77.0 ± 0.3^b	81.3 ± 0.6^b	38.9 ± 0.6^a	4.7 ± 0.5^a
PSP sample	4.1 ± 0.2^b	41.6 ± 0.3^a	53.1 ± 0.2^a	64.5 ± 0.5^a	47.7 ± 0.8^b	8.5 ± 0.4^b
Note: The values were expressed as mean ± S.D. of 3 assays. Values with the same lower case superscript letter within a column are not significantly different (p < 0.05) as determined by analysis of variance (ANOVA).

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. DSC data (Table 1) indicated that in the process used in this study, pressurized high temperature cooking realized full gelatinization of the raw dried peas and broke the original crystal structure of the pea starch, which resulted in disappearance of the original heat-absorption peak (Tp, 77.0°C) from the processed PSP product. However in the processing steps afterwards, amylopectin molecules rearranged and re-crystallized, and a retrograde peak appeared in DSC analysis (Tp, 55.4°C); The same result was observed from the retrogradation of waxy rice starches, their retrogradation peak temperature were about 50–53°C, while the native starches' were at 56–75°C (16). Compared with the natural crystals in the raw material, the crystals content was higher in processed PSP product as evidenced by the higher phase transition enthalpy (δH) with 4.1 J/g. In addition, Table 1 shows that after pea processing, its δH increased in parallel with its content of RS and SDS, which suggested that the decreased digestibility of the final PSP product may be attributed to retrograded amylopectin.

Influence of Processing Conditions on Digestibility of the PSP Product

Based on the results of the above experiments, we further studied the influence of different processing conditions on the digestibility of the PSP product (Fig. 3). Fig. 3A shows that in the pressurized cooking step, material: water ratio of 1:6 gave the highest RS yield and lowest SDS yield while higher or lower material: water ratios were not conducive for RS formation. 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.

Figure 3 Effect of processing conditions on the digestibility of PSP products. A: cooked at 121°C for 3 h; soaked for 2 h; normal heat dehydrated for 30 min; B: cooked for 3 h with material/water ratio of 1:6; soaked for 3 h; normal heat dehydrated for 30 min; C: cooked at 121°C with material/water ratio of 1:6; soaked for 1 h; normal heat dehydrated for 30 min; D: cooked at 121°C for 3 h with material/water ratio of 1:6, normal heat dehydrated for 30 min; E: cooked at 121°C for 2.5 h with material/water ratio of 1:6; soaked for 1 h; after 30 min normal heat dehydrated, continued to stir-heated till 45 min.

The yield for RS increased as cooking temperature increased, and the highest yield was at 121°C (Fig. 3B); in contrast, the content of SDS peaked at 111°C. Amylose-lipid complexes dissociate normally at 95–100°C to release amylose molecules.[17] High temperature and high pressure treatment would result in full gelatinization of starch granules and complete migration of amylose molecules. Additionally, the higher cook temperature the lower the viscosity of the resultant starch paste and the reduced viscosity conditions free starch. And under lower viscosity, free starch (especially amylose) molecules have easier access to each other and tend to form inter-molecular hydrogen bonds, which is conducive to full association of amylose double helix molecules and the formation of RS. However, too high a process temperature will cause further degradation of starch molecules to yield low molecular weight hydrolyzates with limited potential for re-associations. Fig. 3C shows that the yield of RS first slightly increased and then declined as cooking time increased from 2 to 3.5 h but the slope of change was rather flat and the differences may not be significant.

Figure 3D shows that RS content was highest with 5 h soaking while SDS level was highest with 24 h soaking. Retrogradation is a phenomenon dramatically different from gelatinization, i.e., gelatinized starch spontaneously changes to natural-starch-like water-insoluble state over time. 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; 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. Our results were different from the report by Eyaru et al.,[18] which contributed to the different consequence of soaking and cooking. They soaked the peas before cooking, and this resulted in reduced starch fractions, possibly due to leaching of soluble fractions.

Figure 3E shows that RS content increased while SDS content decreased quickly as heat dehydration time increased from 30–32 min and the levels became stabilized beyond 32–33 min heat dehydration time while the content of RDS remained unchanged. It is evident that SDS was converted into RS during heat dehydration. It is possible that during heat dehydration some imperfect crystal SDS underwent structural rearrangement to a more orderly structure and became RS, which has extensive crystallization. Meanwhile, it is also possible that the heat dehydration process changed the physical structure of the SDS such as crystal structure and increased its density which produced more low digestible starch species, i.e., RS.

Under different processing conditions, SDS and RS are significantly correlated (2-tailed correlation analysis) (Table 2), indicating that various processing conditions promote the inter-conversion between RS and SDS. Therefore, processing conditions can be changed to effectively control the relative content of SDS and RS in PSP products. This methodology may enable process modifications to influence the functional digestibility properties of prepared PSP products.

Table 2 Correlation between starch content under different processing conditions

Correlation between	r	p
RDS and SDS	0.433	0.02*
SDS and RS	0.983	0.00**
RS and RDS	0.590	0.01**
RDS: rapid digestible starch; SDS: slow digestible starch; RS: resistant starch. The correlation analysis was done between each two different digestible starch using their contents in the total amount of PSP product; **: significant at the level of p < 0.01; *: significant at the level of p < 0.05.

CONCLUSIONS

Changes in processing conditions for PSP product had minimal impact on the content of RDS, but specifically affected the content of SDS and RS. Results inferred 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.

ACKNOWLEDGMENT

This article is supported by 111 project-B07029 and PCSIRT0627.