Reduction in soaking time and anti-nutritional factors by high pressure processing of chickpeas (2024)

Material

Dried Canadian Kabuli chickpeas (CLIC brand) packed in a heat-sealed clear plastic polyethylene bags weighing 407g per packet were purchased from a local supermarket (Provigo Distribution Centre, Montreal) and stored at room temperature. Before using, chickpeas were washed thoroughly in water, and damaged, spotted and split pieces were removed.

Sample preparation

About 10 pieces of dried chickpeas was accurately weighed (~ 0.4g) with a precision balance (± 0.0001g accuracy) (APX-200 Digital Weighing Balance, Denver Instruments, USA) and placed in 7oz. low-density polyethylene bag (Whirl-Pak(R), Nasco, Fort Atkinson, WI, USA). The bag was then filled with 25mL of distilled water (conductance: 18V, Milli-Q, Millipore, Bedford, USA) and heat sealed and transferred to the HP equipment for treatment.

High pressure treatment

High pressure treatments were given in an HP equipment (ACIP 6500/5/12VB-ACB Pressure Systems, Nantes, France) consisting of a cylindrical pressure chamber of 5L volume. The pressure–time (P–t) treatment was administered using a computer connected to a data logger (SA-32, AOIP, Nantes, France). The pressure transmission medium used was water. The pressurization rate was 5MPa/s up, upon reaching the target pressure, a 10min treatment (holding time) was given followed by a rapid depressurization (< 4s) to atmospheric pressure. Samples were divided into 2 groups: a) the first group samples were HP treated without pre-soaking and while the second group samples were pre-soaked for 2h at 40°C before HP treatment. Six HP treatments were given at: 100, 200, 300, 400, 500 and 600MPa, and at each pressure, a single pressure cycle (pressure come-up, 10min hold, depressurize) or up to six such pressure cycle treatments were given. In addition, a single 20min holding time pressure cycle was also included to compare with two 10min holding time cycles (same total holding times but with one or two cycle pressure treatments). After HP treatment, the samples were analyzed for various quality parameters.

Water absorption

Water absorption capacity of chickpea samples were measured according to Yu et al. (2017). For this specific purpose, individual chickpea samples were weighed and packed in separate plastic bags and HP-treated. Following HP treatment, chickpeas with and without pre-soaking (2h at 40°C) were removed from the plastic bags and wiped with a wet towel to remove the excess water on the surface and then weighed. Water absorption of HP treated samples were computed using Eq.(1) and compared with samples soaked at room temperature and at 40°C for 3, 6, 9, and 12h. All measurements were carried out in triplicate. Water absorption or hydration capacity (wet basis) was calculated according to following equation:

where WA (%) is the water absorption percentage of the sample (wet basis), M₀ is the mass of dry sample without treatment (g), and M_t is the mass after treatment either HP or regular soaking (g) treatment for a specific time (t).

Color

Color parameters of raw chickpeas (control), chickpeas soaked overnight at room temperature and HP treated samples with and without pre-soaking were determined in the L, a, b system using a tristimulus Minolta Chroma Meter (Minolta Corp., Ramsey, NJ, USA). The instrument was warmed up for 5min then calibrated with a white standard prior to use. Ten measurements were taken with each sample in order to obtain the average values of L (lightness), a (green (−) to red (+)) and b (blue (−) to yellow (+)). The ΔE (total color change) was also determined according to the following equation:

where L₀, a₀, b₀ represent the values of dry chickpeas and L, a, and b represent HP treated samples.

Texture profile analysis

Texture properties of raw and HP treated chickpeas were evaluated using a TA.XT.Plus Texture Analyser (Texture technologies corp., Scarsdale, NY, USA) previously calibrated with a 50kg load cell, a fixed platform and a 25mm diameter cylindrical probe. The samples were uniaxially compressed at room temperature to 80% of their original height. Each sample was subjected to two subsequent cycles (bites) of compression–decompression. The crosshead speed was set to 5mm/s for the first and the second bites, respectively. Canned chickpeas samples were analyzed as a reference for commercial level of product texture. The instrument computed different parameters through the software such as hardness (maximum force required to compress the sample), adhesiveness (work necessary to pull the compression anvil away from the sample), chewiness (gumminess × springiness), cohesiveness (area 2/area 1), gumminess (hardness × cohesiveness), and springiness (area a/area b). The target parameters used for this study were hardness and chewiness because they reflect the quality of chickpeas during mastication (Cardello and Segars 1989). The analysis was performed with ten replicates and the average shown in the results. The instrument automatically recorded the force–displacement and converted them to texture profile analysis.

Scanning electron microscopy

The microstructure of HP treated sample without pre-soaking at 200, 400, and 600MPa for 20min, overnight soaked samples at room temperature and raw dry chickpea samples were examined after lyophilization through a scanning electron microscope (SEM) (Hitachi Tabletop Microscope, TM3000). SEM was performed to investigate the effect of HP on pores (cavities) and the increase in starch sizes (Cappa et al. 2016; Yu et al. 2017). Cell sizes were determined using image processing software for scientific analysis (ImageJ 1.50i). Each sample was placed on a carbon layer which was in turn stuck to a rotary holder before being scanned and photographed at 500X magnifications.

Phytic acid content

Phytic acid content was measured according to McKie and McCleary (2016) using a Megazyme phytic acid (Phytase/Total phosphorus) assay kit (#K-PHYT, Megazyme International Ireland). The assay’s principle is based on the hydrolysis of phytic acid by phytase as well as alkaline phosphatase into myo-inositol (phosphate) and inorganic phosphate (Pi). After that, Pi reacts with ammonium molybdate, which is reduced later to molybdenum blue in acidic conditions. Absorbance of molybdenum blue at 655nm measured with UV/VIS spectrophotometer (VWR, Model V-3100PC) is proportional to the amount of Pi presents in the sample.

Tannin content

Tannin was evaluated using the method described by Khandelwal et al. (2010). The principal was based on reacting condensed tannins with vanillin in the presence of acid to produce red color. One gram of high pressure (HP) treated chickpeas from both groups (with and without pre-soaking), soaked overnight samples at room temperature and dry chickpeas were extracted with 20mL of 1% HCl (ACROS ORGANICS, NJ, USA) in methanol (LC–MS Grade, EMD Millipore Corporation, USA) for 20min in water bath at 30°C. The samples were centrifuged at 2000rpm for 4min. The supernatant (1mL) was reacted with 5mL vanillin solution [0.5% vanillin (99% pure, ACROS ORGANICS, NJ, USA) + 2% HCl in methanol] for 20min at 30°C. Blanks were run with 4% HCl in methanol in place of vanillin reagent. Absorbance was read at 500nm on a UV/VIS spectrophotometer (VWR, Model V-3100PC). A standard curve prepared with catechin (TRC Canada, Toronto, ON, Canada). Tannin content was expressed as mg CE/100g. Samples were analyzed in triplicate.

Solid loss determination

Percent of solids lost after soaking overnight or after HP treatment was determined by drying a known amount of the soaking water at 105°C until constant mass is reached. The percentage of solid loss (SL %) was calculated from the following equation:

where M_L and M₀ are the total amounts (g) of solids in the soaking water, and in the raw seeds, respectively.

Fourier transform infrared (FTIR) spectroscopy

The FTIR spectra of dry chickpeas, room temperature overnight soaked chickpeas and HP treated chickpeas samples with and without pre-soaking were obtained by using a Manga System 550 FT-IR Spectrometer (Agilent 5500a, Northen ANI, Solution, USA) over a wavelength range of 400–4000cm⁻¹ equipped with an OMNIC operating system software (Version 7.3, Thermo Electron Corporation). FTIR has been used previously to observe oligosaccharides reduction (Yoshida et al. 1997). Samples were covered on the surface in contact with attenuated total reflectance (ATR) on a multi-bounce plate of Zn-Se crystal at 25°C. All spectra were background corrected using an air spectrum, which was renewed after each scan. Each spectrum was collected from an average of 32 scans with a resolution of 4cm⁻¹ and the results were reported as mean values.

Statistical analysis

Statistical analysis was performed by using SPSS software version 20. Data were expressed as means of at least triplicate and analyzed by a one-way analysis of variance (ANOVA). p value ≤ 0.05 was regarded as significant based on Duncan’s multiple range tests.

Water absorption

Chickpeas hydration was evaluated before applying HP treatment by soaking them at room temperature and 40°C for 3, 6, 9 and 12h. Results showed hydration percentages were 53.1 ± 3.06, 81.8 ± 1.77, 82.7 ± 1.96, and 83.9 ± 1.34, respectively, after 3, 6, 9, 12h soaking at room temperature and 70.7 ± 3.24, 84.2 ± 1.98, 84.1 ± 3.56, and 84.4 ± 0.78, respectively at 40°C. These results were used for comparing the soaking performance of HP treated chickpeas.

Figure1a compares the hydration capacity of single cycle 20min vs two-10min-cycle hold time HP treatments. A significant difference in percentage hydration was observed between the two HP treatments at each pressure level employed between 100 and 600MPa for chickpea samples without the pre-soaking treatment. The two-cycle action resulted in higher hydration than the single cycle treatment (with same total hold time), possibly because of the additional structural disturbance due the two compression-decompression actions in the two-cycle treatment. Hydration of chickpeas during soaking or during the soak-pressure treatment results from the absorption of water and results in swelling of starch granules or imbibition of water by proteins. With soaking treatment of brown rice, Yu et al. (2017) found the opposite results probably because of the difference in the nature and composition of the samples: brown rice vs. chickpeas, which differ in their carbohydrate and protein contents [carbohydrate (78% & 67%) and protein (7% & 20%) in rice and chickpeas, respectively]. Higher protein content in chickpeas influences water retention in moderate pressure intensities (200–500MPa) since protein aggregation happens which allows more water to be retained in the cavities. Comparing the hydration at different pressure levels, 400–500MPa treatments resulted in higher hydration difference than lower pressures (200 and 300MPa). These results lead to the importance cycles and to further explore additional cycles and added pre-soaking treatments.

Figure1b illustrates hydration capacity of multiple cycle HP treated samples chickpea at different pressure levels, again without any pre-soaking. Moisture uptake ranged from 23 to 88% with six consecutive HP 10min cycles. Single cycle treatment showed different hydration rate at each pressure level as well as each of the multiple cycle treatments, clearly showing a dependence on both pressure level and number of cycles. Higher intensities resulted in higher hydration capacities ranging from about 25 to 40% as the pressure level increased from 100 to 600MPa. The increased hydration efficiency at higher pressures could be because HP could breakdown cellular structure and allow more free water to penetrate the cell. Also, hydrogen bonding formation between water molecules and starch granules could help in increasing hydration process (Yu et al. 2016; Xue et al. 2018). Until cycle number six, there was a continuous increase through all pressure intensities except for 500 and 600MPa that showed a slight decrease that might be due to structural change and rupture of the cell wall that will be discussed in the following sections.

Figure1c shows the influence of pre-soaking (2h at 40°C) treatment on the hydration capacity of chickpea samples following HP treatment at different pressure levels (100–600MPa) and with up to six consecutive cycles 10min each. Pre-soaking resulted in an average of 70% (± 4.2%) hydration. After HP treatment, the highest increase in moisture content was during the 1st cycle 17% (± 3.0%) followed by slight increase which did not exceed 4% (± 1.2%) during 2nd cycle only. Further cycles displayed a fluctuation in hydration rate and ended up with a general reduction in hydration by 2–7% between 200 and 600MPa. It is clear from Fig.1c that the maximum hydration was ≈ 90–93% for almost all intensities which means that samples were fully hydrated at that point. The reason of moisture reduction in almost all cases with the highest at 600MPa is because pores (cracks) were caused by HP that led to losing of some water after full hydration plus increase in soluble solids extraction (Sayar et al. 2001; Yu et al. 2016). Turhan et al. (2002) showed that chickpeas could reach the maximum hydration ≈ 105% after 10h soaking at room temperature. In this study, the maximum hydration was reached with 20min HP treatment after 2h pre-soaking or 50min treatment without pre-soaking.

Color change

High pressure treatment of chickpea with and without prior pre-soak treatment (2h at 40°C) affected color values significantly at different pressure levels (100–600MPa) and pressure cycles (1–6) with a 10min holding time (Table1). The control samples for treatments with and without prior pre-soak has significantly different color values from treated samples.

The L values (brightness) were higher for pre-soaked samples than those for samples without pre-soak treatment and they increased linearly with both treatment pressure (brighter color) and pressure cycles. The increase in L could be due to possible surface migration of white endosperm materials, increase in sample hydration or removal of entrapped intercellular gases. Hydration has been considered as the main reason in earlier studies (Yu et al. 2017; García-Parra et al. 2016; Tian et al. 2014).

The a values (redness) of controls and HP treated chickpea samples also demonstrated significant differences. Generally, the control samples had lower a values than treated samples. Redness was the highest in the first cycle then decreased significantly in the second one and stayed stable throughout the additional cycles. Overall, a values of samples without pre-soaking were higher than after pre-soaking. García-Parra et al. (2016) and Tian et al. (2014) studies support these results. The reason of lowering a* with HP treatment as explained by Saikaew et al. (2018) was due to cell rupture which causes enzymatic and non-enzymatic reactions in corn samples.

Yellowness (b) was the lowest in both controls and then increased significantly through the 1st cycle of high pressure (HP). After that they decreased until 3rd or 4th cycle then returned to increase slightly. Overall yellowness was higher in samples that were not pre-soaked. Yu et al. (2017) findings support the decrease b values and reasoned that the reduction may be due to the diffusion of yellow pigments from brown rice samples into water during high pressure treatment.

To evaluate the overall color change, ∆E values were also compared. Single cycle HP treatment at 100MPa with and without pre-soaking were 6.71 (± 1.08) and 17.9 (± 0.53), respectively, demonstrating a large increase in ∆E. Color change after HP treatment for samples without pre-soaking were sharper than in pre-soaked ones. Extreme treatment conditions (600MPa, 6 cycles) resulted in ∆E 31.3 (± 0.75) and 29.9 (± 2.85) with and without pre-soaking indicating that the differences could be reduced with HP treatment intensity. However, there was a huge ∆E difference between the minimum and maximum HP treatment. It was clear that ∆E increased with increasing pressure intensity for both categories. García-Parra et al. (2016) also found that higher pressures resulted in larger ∆E than lower pressures for pumpkin.

Texture profile analysis

Scanning electron microscopy (SEM)

Scanning electron micrographs of chickpea samples soaked overnight and treated with 0.1, 200, 400 and 600MPa for 20min are illustrated in Fig.3 (500× and 250×). Starch granules were oval and round-shaped in untreated and overnight soaked samples. HP treated samples retained the same oval and round shape, but with swelling as also observed by Ahmed et al. (2016a). An average of 0.34, 0.38, 0.38, 0.37 and 23.88μm for raw chickpeas (0.1MPa), soaked overnight, treated with 200, 400, and 600MPa, respectively. So, a significant change in granule diameter was observed with all samples. The main purpose of soaking is to help absorption of water which facilitates easy gelatinization of starch during cooking. It can be achieved either through conditioning below the gelatinization temperature then cooking or through direct cooking in water above the gelatinization temperature. During high pressure treatment, water is forced into chickpea samples and causes more rapid hydration which is driven by the external pressure applied. This results in rapid swelling even at ambient temperatures. Swelling is a result of the increase in hydrogen bonding between water and starch which promotes water uptake (Yu et al. 2016; Turhan et al. 2002; Sayar et al. 2001). Raw and overnight soaked samples showed compact tissues. On the other hand, HP treated samples showed bigger pores sizes and aggregated granules especially for samples treated with 600MPa. Those aggregates might explain the reason for the relatively harder texture that was discussed previously. Big pore sizes were the reason for enhanced hydration and swelling of granules due to easily water diffusion. Surface and cell wall of overnight soaked samples was compact and intact compared to 600MPa treated one. Cell wall of 600MPa treated sample was almost disappeared and destroyed with very big size pores that proves the damage cause by HP (Denoya et al. 2016; Yu et al. 2017; Yu et al. 2016; Ahmed et al. 2016b).

Fourier-transform infrared spectroscopy (FTIR)

FTIR is an important technique to observe whether any change happened to the structure of the sample when applying any processing such as pressure, thermal treatment, chemical treatment or any other types of treatments. Figure4a illustrates spectrum of HP treated chickpea samples, soaked overnight and raw chickpeas in the spectral region 2800–3700cm⁻¹. Control (soaked overnight) sample and HP-treated samples showed similar main peaks with just a little difference in the amplitude of peaks which confirmed that all of them absorbed water but with different levels.

On the other hand, raw sample differed considerably in the major peak at 3276cm⁻¹ in which it had lower intensity compared to HP treated samples with a higher intensity peak at 2923cm⁻¹ and an extra peak at 2853cm⁻¹ that was absent for other samples. Higher intensities are an indicative of crystallization suggesting strong hydrogen bonding whereas the absence of the band showing an amorphous structure which indicated the effect of HP on chickpeas carbohydrates structure (Wolkers et al. 2004). The reason of the difference between raw and processed samples was the degree of crystallinity by relatively sharp absorption bands shown in raw sample, whereas broader absorption bands (2932cm⁻¹) were visible in amorphous HP treated chickpeas. Crystalline structure had also a shoulder peak in the OH stretching region that was around 3000cm⁻¹ which is an indicative of hydrogen bonds (Wolkers et al. 2004).

Figure4b is mainly focused on carbohydrates with the carbohydrate fingerprint region 900–1200cm⁻¹. A considerable difference between raw sample and HP treated ones can be noticed. Like previous figure, raw chickpeas had crystalline structure than HP treated samples since it contained sharper and higher peaks’ intensities. Present results of overall carbohydrate peaks shapes of chickpeas are supported by Sun et al. (2014). Wolkers et al. (2004) reported that bands’ shift to lower wavenumbers means dehydration in addition to broader peaks which is proved by present results having almost all peaks of raw dry chickpeas broad and shifted to lower wavenumber. Major bands absorption at 994cm⁻¹ shifted to around 1000cm⁻¹ with increasing pressure which is an indication that the crystalline structure of starch disappeared through HP treatment since HP affects starch gelatinization (Ahmed et al. 2016b). The signal at 1740cm⁻¹ in raw chickpeas represented vibrations of ester groups in pectin. Since this peak is absent in HP treated samples, then it might be either the cell wall destroyed with HP or an overlap took place with the strong water band centered at 1640cm⁻¹ (Chen et al. 1997). It was reported that peaks at 1146cm⁻¹ and 1100cm⁻¹ are assigned to the PO₂ stretching modes, the P–O–C anti-symmetric stretching mode of phosphate ester, and to the C–OH stretching of oligosaccharides. The decrease in intensity of 1146cm⁻¹ peak was attributed to the decrease in oligosaccharide content (Yoshida et al. 1997). As a result, raw chickpeas had higher oligosaccharides than pressurized and overnight soaked samples.

Phytic acid and tannin contents and solids loss

Table2 shows the results for phytic acid and tannin content and solid loss in selected HP treated samples to compare the quality of samples after HP treatment as compared to overnight soaked samples and raw dry chickpeas. Soaking overnight reduced phytic acid content significantly to almost one third of that in the raw sample. HP treated samples reduced the phytic acid levels to more than overnight soaked samples. The most powerful way of reducing the phytic acid content was HP processing of pre-soaked samples at 500MPa for two consecutive cycles. Soaking prior to HP treatment is advantageous from phytic acid reduction point of view. Other studies (Deng et al. 2015; Linsberger-Martin et al. 2013) have shown the significant effect of HP treatment on phytic acid reduction in beans, peas and buckwheat compared to untreated samples. The reduction might be attributed to the hydrolytic activity of the enzyme phytase which gets activated during soaking (Deng et al. 2015; Sinha and Kawatra 2003).

Overnight soaking reduced tannins content of raw chickpeas by more than half and as observed with phytic acid (Table2); HP treatment contributed to further lowering up to 24% of the initial level. Among the 4 samples treated with HP, the one treated with 200MPa for 6 consecutive cycles reduced tannin content slightly more than the other 3 samples. Deng et al. (2015) reported that HP processing at 600MPa for 30min could reduce tannins by about 20%. The difference might be due to the use of multiple cycles in the present study which helped to reduced tannins to a greater extent. General reason of tannin reduction is that they are water soluble, so they leach out into the water (Uzogara et al. 1990).

Solids loss is another quality parameter that should be taken in consideration since part of the present study was to better understand the HP treatment for hydration of chickpeas. Solids loss and water absorption can occur simultaneously during any soaking treatment. When water penetrates chickpeas, it can solubilize some carbohydrates and proteins in addition to causing some vitamins and minerals to leach out into water (Sayar et al. 2009). The lowest solid loss was associated with overnight soaking. Since no literature could be found to compare HP effect on solids loss, comparison of normal soaking between present study and other studies was done. 2.2% was the solid loss for 12h soaking at 25°C in present study versus 2.5% for 15h at 20°C in a study conducted by Sayar et al. (2009) and 0.7% for 6h at 25°C in another study (Johnny et al. 2015). Both studies were on Kabuli chickpeas.

Solids loss in HP treated samples were significantly higher than in overnight soaking except for the one treated with 200MPa for 6 cycles. It had a solids loss average of 2.4%. Generally, HP processed samples that were pre-soaked had significantly higher solid loss percentage than the ones without pre-soaking regardless pressure intensity or number of cycles. It is logical because presoaking and high pressure considered as two soaking steps rather than one in HP processing. In addition, pre-soaking temperature was 40°C and it is known that the higher the temperature the more solid loss (Johnny et al. 2015).

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