Application of High Hydrostatic Pressures and Refrigerated Storage on the Content of Resistant Starch in Selected Legume Seeds

Abstract

Resistant starch (RS) is a fraction of starch not digested and absorbed in the small intestine, and it is fermented by the intestinal microbiota in the colon, thereby influencing many health benefits. Legumes such as beans, lentils, and chickpeas are rich in fermentable dietary fiber, and RS can be included in this fiber group. These legumes are not considered a “typical” source of starch and have not been extensively studied as a source of RS. There are still insufficient data on modern non-thermal methods like high-pressure processing (HPP) and combining this method with refrigerated storage. The study aimed to investigate and compare the effects of HPP and HPP combined with refrigerated storage on the RS content of legumes, particularly white beans, green lentils, and chickpeas. Different pressure levels and processing times were used to evaluate changes in RS content and to assess the total fiber content and fiber fraction of the tested legumes. Our study showed that the increase in pressure and pressurization time affected changes in the RS content of the examined legumes. Furthermore, the cooling process of previously pressurized samples resulted in a significant increase in RS content.

1. Introduction

Legume seeds constitute a precious source of essential nutrients, including proteins, RS, minerals, and vitamins [1]. Lentils (Lens culinaris), beans (Paseolus vulgaris), and chickpeas (Cicer arietinum) stand out as the most popular and widely consumed legume seeds globally [2]. Numerous studies have underscored the health benefits associated with the consumption of legumes. Besides being rich in plant proteins and fiber and having a low glycemic index (GI), legume seeds are also a notable source of starch, particularly in the form of RS [3,4,5].

The gut microbiota can ferment various fractions of dietary fiber and resistant starch. The fermentation process in the colon involving the microbiota contributes to the production of beneficial short-chain fatty acids (SCFAs), such as butyrate, acetate, and propionate. These metabolites play an essential role in maintaining the integrity of the intestinal barrier, modulating inflammation, and influencing the metabolism of many nutrients [4,5,6].

RS is characterized as the fraction of starch that escapes digestion in the small intestine, reaching the colon, where it undergoes fermentation with the involvement of intestinal microbiota. RS can be categorized as a fermentable dietary fiber characterized by low viscosity and solubility [6]. However, quantifying RS is acknowledged as a challenging task due to variations in hydrolysis conditions, incubation terms, method selection, and enzyme concentrations [7,8]. Numerous in vitro and in vivo studies have demonstrated that the consumption of RS yields positive effects on postprandial glycemic response [9,10], enhances insulin sensitivity [11,12], and reduces inflammation [13,14,15]. While recent research has predominantly focused on starch transformations in potato and cereal starch [16,17,18,19,20,21,22], there is a noticeable dearth of studies examining the nutritionally valuable legume seed starch. Both thermal and non-thermal processing induce structural changes in starch through gelatinization [5,23]. The degree of starch gelatinization influences the properties of starch and the methods and parameters of processing [5,24,25].

Processing technologies such as high temperature [23], microwave heating [24], or high pressure can cause starch gelatinization [25,26]. Compared with HPP, thermal techniques can lead to the complete disruption of starch granules, resulting in gel formation during extended storage [27]. Thermal methods compromise the matrix and induce starch gelatinization, which can lead to the loss of valuable properties such as vitamins, antioxidants, and flavors, thereby diminishing the product’s health and sensory value. Conversely, non-thermal methods like HPP can alter the structure of large molecules such as proteins and polysaccharides, enabling the inactivation of microorganisms and enzymes while leaving smaller molecules like vitamins, polyphenols, and flavors unaffected [28,29,30]. Consequently, HPP-treated products are characterized by their high sensory quality and associated health benefits [30]. HPP has been utilized to gelatinize or physically modify various starch dispersions, allowing starch granules to undergo gelatinization at room temperature. Previous reports indicated that high-pressure-induced gelatinization of starch depends significantly on the type of starch, water content, pressure, temperature, and processing time [31,32].

However, conclusive studies about HPP’s impact on the starch gelatinization process and RS content in legume seeds are still lacking. Additionally, there is insufficient information regarding the intensity of the retrogradation process in starch products processed with HPP during refrigerated storage. In our study, we selected popular starchy foods from the typical carbohydrate group, like groats, rice, and legumes, to examine total fiber levels. Based on their highest fiber content, we selected three legumes for further study. The study aims to compare the impact of HPP and HPP combined with refrigerated storage on RS content in legumes, specifically white beans, green lentils, and chickpeas. Various pressure levels and processing times were employed to assess changes in the RS contents and evaluate total fiber and fiber fractions in the tested pulses.

2. Materials and Methods

For the initial analysis of dietary fiber content, starchy products from different food groups, widely available and consumed in Poland, were selected, as shown in Figure 1. Standard carbohydrate products were implied, but a group of legumes was also chosen to compare the fiber content. Such products as brown rice, bulgur groats, millet groats, green lentils, red lentils, peas, beans, and chickpeas were selected. An analysis of the fiber content was carried out, and based on this, three products with the highest fiber content were selected for further analysis. Legume seeds, including white beans, chickpeas, and green lentils, were utilized for this study. All dry-form seeds were purchased at a grocery store in Warsaw and came from Melvit S.A. (Warsaw, Poland). The Resistant Starch Assay Kit for the measurement and analysis of resistant starch in plant materials and starch samples was bought from Megazyme International Ireland (Bray Business Park, Bray, Co. Wicklow, Ireland). The following official analysis methods were used: AOAC Method 2002.02, AACC Method 32-40.01, CODEX Type II Method. All chemicals, standards, and reagents were of analytical grade, and reputable suppliers provided other materials. The seeds were ground in an analytical electric grinder (Rommelsbacher EKM 100, Rommelsbacher ElektroHausgeräte GmbH, Dinkelsbühl, Germany), sifted through a 1 mm sieve (Labindex S.C., Warszawa, Poland), dried, and then packed in airtight glass containers with a closure.

The Gravimetric (dryer-weight) method determined the dry matter content at 105 °C [33]. Dietary fiber content was measured using the AOAC 991.43 method. The samples underwent sequential treatment with the following enzymes: alpha-amylase, protease, and amyloglucosidase. Subsequently, ethanol precipitated the soluble fiber, separating the protein and glucose from the samples. The residue was filtered, successively washed with ethanol and acetone, and then dried and weighed. One duplicate was analyzed for indigestible protein and the other for ash. Dietary fiber represents the residue after analysis adjusted for indigestible protein and ash content.

Starch-water solutions (20%, w/w) were prepared at room temperature and hermetically sealed in high-density polyethylene containers. The packaged samples were placed into a cylindrical loading chamber and subjected to high hydrostatic pressures at 200, 400, and 600 MPa for 3, 5, 6, and 9 min at room temperature (25 °C) in an HPP chamber (EXDIN Solutions Ltd., Kraków, Poland), with water as the pressure transfer medium. The time to reach the determined pressure was less than 90 s, and the decompression time was less than 15 s. Following the process, a portion of the samples was directly analyzed for RS content. In contrast, the remaining portion was stored under refrigeration (4 °C) for 24 h before undergoing the same analysis. Each analysis was carried out in triplicate.

RS analyses were performed using the AOAC 2002.02 method. Samples underwent incubation and shaking with pancreatic α-amylase and amyloglucosidase (AMG) in a water bath at 37 °C for 16 h. The non-resistant starch was dissolved and hydrolyzed to D-glucose through the combined action of the two enzymes. The reaction was halted by adding an equal volume of ethanol or methylated industrial spirit (IMS, denatured ethanol), and the RS was recovered in pellet form during centrifugation. Subsequent steps included two washes by suspension in aqueous IMS or ethanol (50% v/v), followed by centrifugation. The free liquid was removed through decanting. The granular RS was then dissolved in 2 M KOH and stirred vigorously in an ice water bath with a magnetic stirrer. The solution was neutralized with acetate buffer, and the starch was quantitatively hydrolyzed to glucose using AMG. D-glucose was measured with glucose oxidase/peroxidase (GOPOD), and RS content was measured in the sample. A light microscope (DM-400M-LED, Leica, Wetzlar, Germany) was used to observe changes in the starch samples. One drop of the suspension was applied to a microscope slide and covered with a coverslip. Polarized light mode and 600× magnification were used for imaging.

All statistical analyses were performed using Statistica, version 13 (TIBCO Software Inc., Palo Alto, CA, USA). The accepted level of statistical significance for evaluating differences in mean RS content was a = 0.05. Descriptive statistics were performed for all cases by calculating mean values and statistical deviations. The significance of the differences in the mean content of RS in samples without cooling and in cooled samples was tested using a one-way analysis of variance. For each plant, the effect of applied pressure (at constant time) and pressurization time (at constant pressure) on RS content were evaluated. Once a statistically significant F-test value was obtained, Tukey’s (HSD) tests were performed to determine which averages were significantly different from each other. The effect of cooling on the average RS content of each plant was evaluated by conducting an analysis of variance with repeated measurements.

3. Results and Discussion

3.1. Legumes as a Source of Dietary Fiber

Analyses of the total fiber content in legume seeds revealed that the highest values were obtained in white beans (20.46% dry matter), followed by chickpeas (14.7%), with the lowest value found in green lentil seeds (13.75%). Additionally, the results indicated that both beans and lentils, along with chickpeas, exhibited a higher content of the insoluble fiber fraction compared with the soluble fraction.

According to recommendations from international organizations, a daily dietary fiber intake of 14 g/1000 kcal or higher can yield measurable health benefits. However, in developed countries, there is still an inadequacy in daily fiber intake [34,35]. The expansive dietary fiber category encompasses numerous components, each with distinct uses and health benefits. In recent years, fractions such as arabinoxylan, inulin, β-glucan, and RS have garnered increased attention [6]. Incorporating legume seeds into daily meals can be an effective strategy for meeting fiber needs, particularly in highly developed countries, as the total fiber content of legume seeds can reach up to 30 g/100 g dry weight [36]. Currently, many recognized functions of dietary fiber are related to metabolic health, primarily in lipid and carbohydrate metabolism. However, there has been growing attention in recent years to the effects of different fiber types on the gut microbiome [37,38,39,40]. While the number of studies on fiber is expanding, most still focus on cereal products and tubers, neglecting pulses as a fiber source [41,42,43,44,45]. Despite the increasing research on fiber composition in foods, broadening the study of legumes for this prebiotic nutrient is crucial. There are no official guidelines on which sources and fractions of dietary fiber are essential for improving the gut microbiota [34,46,47,48,49]. The content of total dietary fiber and its fractions—soluble and insoluble—can vary within a single product group due to factors such as botanical origin, cultivation conditions, region of origin, harvesting, technological processing, and the research analysis method used [50]. In our study of total fiber and its fractions, the highest values of total fiber were obtained in white beans (20.46%, dry weight), followed by chickpeas (14.7%), and the lowest value was found in green lentil seeds (13.75%), which has also been supported by other researchers [46,51,52,53]. Results from our study (Figure 2) and studies by different authors confirm that legume seeds such as beans, lentils, and chickpeas are characterized by a predominance of the insoluble fraction of fiber over the soluble fraction, regardless of the analytical method used [46,51,52,53,54]. Considering that the described fiber values in legumes are very high compared with other foods, increasing the consumption of legumes appears reasonable as it may positively impact replenishing fiber deficiencies, especially in developed countries [36,48,49].

3.2. Effect of HPP on RS Content

3.2.1. Effect of Pressurization Time and Pressure Value on RS Concentration

RS content was analyzed in unprocessed samples, which were the control samples. The results showed RS contents of 0.96 g/100 g, 1.99 g/100 g, and 1.53 g/100 g, respectively, for beans (Figure 3), chickpeas (Figure 4), and lentils (Figure 5). All RS results were presented in grams/100 g of sample. Statistical analysis of the averages for uncooled bean samples showed that the average RS content for the control sample (unprocessed starch) and samples pressurized at 200 and 400 MPa were significantly higher than the average at 600 MPa (Figure 3b). On the other hand, pressure time in uncooled samples resulted in a decrease in average RS values compared with the control sample (Figure 3a). Meanwhile, a pressure time of 5 min in cooled bean samples resulted in a substantial reduction in RS compared with the control sample, with times of 3, 6, and 9 min (Figure 3c).

In the case of chickpeas, statistical analysis of the averages of RS content in uncooled samples treated at 200, 400, and 600 MPa showed a statistically significant effect of pressure (p < 0.05). All averages were different at the accepted level of significance. The lowest average RS content was characterized by the sample treated with 600 MPa pressure. The RS content decreased as the applied pressure increased (Figure 4b). A similar observation was applied to the application of variable time (Figure 4a), as RS content decreased with increasing time in uncooled samples, and the lowest mean was characterized by the sample being subjected to 600 MPa pressure. On the other hand, the cooled chickpea samples showed a statistically significant effect of both pressure and pressurization time (Figure 4c,d) (p < 0.05), but the relationship was opposite to that of the uncooled samples—as the pressurization time increased, the RS content increased (Figure 4c). The cooled chickpea samples subjected to increasing pressure showed a decrease in RS compared with the control sample at 200 MPa. Still, applying 400 and 600 MPa pressures resulted in a successively increased RS (Figure 4d).

Uncooled lentil samples treated with pressure times of 3, 5, 6, and 9 min (at a constant pressure of 600 MPa) showed a statistically significant effect of pressure time (p < 0.05). All averages were different at the accepted level of significance. The lowest average RS content was characterized by the sample pressurized at 9 min (Figure 5a). On the other hand, the cooled lentil samples showed an increase in the content of mean RS values during pressurization compared with the control sample (Figure 5c). The analysis also showed a statistically significant effect of pressure of 200, 400, and 600 MPa for both cooled and uncooled samples. In uncooled samples, the average RS values were lower after applying pressures of 200, 400, and 600 MPa compared with the control sample (Figure 5b). On the other hand, for cooled samples, an increase in pressure resulted in a statistically significant increase in average RS values, and all averages differed at the accepted level of significance (p < 0.05) (Figure 5d).

HPP technology in food processing can be applied to starch products as it modifies non-covalent bonds while having minimal effect on the structure of covalent bonds, allowing for the attainment of desired starch properties. Changes in the structure of starch in our study are shown, for example, on the sample of chickpeas treated with 600 MPa pressure for 3, 6, and 9 min (Figure 6). We can see the change in the structure of the starch granules at successive 3, 6, and 9 min (Figure 6b–d) of 600 MPa pressure compared with the unpressurized sample (Figure 6a). The granules of untreated chickpea starch have a spherical morphology, and as the pressurization process is extended, the granules are distorted. Microscopic images showed the enlargement and swelling of HPP-treated starch granules, and we can also see the initial aggregation, which may explain the formation of gel-like granules during the HPP-induced gelatinization process. The results of RS content in the 600 MPa pressure-treated chickpea samples at 3, 6, and 9 min shown in Figure 4a show a gradual decrease in RS content with increasing pressure time, which is probably related to the HPP-induced starch gelatinization process, as can be seen in the microscopic images (Figure 6).

One of the earliest studies investigating the effect of HPP on starch properties dates back to 1981 and focused on the gelatinization of potato starch under the influence of HPP [55]. Since then, more studies have delved into this issue, but the majority have explored “typical” starchy products like cereals and tubers, with limited reports on the effects of HPP on legume seeds as a source of starch [56,57,58,59,60]. In the current study, the RS content changed significantly under the influence of time from both cooled and uncooled chickpeas (Figure 4a,c), beans (Figure 3a,c), and lentil samples (Figure 5a,c). Further, under the influence of pressure, significant changes were observed in both cooled and uncooled beans (Figure 3b,d) and chickpeas samples (Figure 4b,d), as well as in lentil samples (Figure 5b,d). Ahmed et al. (2017) demonstrated no significant differences in the RS content of chestnut flour treated with HPP (0.1, 400, 500, and 600 MPa for 10 min). They indicated that HPP did not cause a change in RS content [61], but in our study, the RS content changed significantly under pressure in the case of all tested plants. In another study by Ahmed et al. (2016), the effect of HPP on the properties of lentil starch under different pressures was studied, and the results showed that a decrease was noted in RS content after applying pressures of 400 MPa [62], which was also confirmed by our study (Figure 5b). However, the authors observed a significant increase in the RS content of lentil starch at a pressure of 600 MPa, which was explained by the rise in temperature during the process due to the adiabatic effect when higher pressure was applied [62]. In our study, the temperature during pressurization did not change, which may be related to opposite observations (Figure 5b). On the other hand, in a study reported by Deng et al. (2014) [63], the effect of HPP on rice starch was investigated. It was shown that higher pressure (600 MPa) resulted in lower RS content to lower pressure (200 MPa), which was also confirmed in our studies, especially with pressure 600 MPa, as depicted in Figure 3b, Figure 4b and Figure 5b.

3.2.2. Effect of Cooling

Research conducted thus far has primarily focused on assessing the impact of cooling following the application of various techniques such as cooking [64], autoclaving [25], and extrusion [65]. However, there is no research evaluating the impact of cooling after prior HPP treatment. The results shown in Figure 7 reveal an increase in RS content for lentils, chickpeas, and beans after the cooling treatment (24 h, 4 °C). After applying all the pressures and pressurization time, we showed the average cooling effect on the tested samples. This increase was found to be statistically significant (p < 0.05). Yadav et al. demonstrated that multiple heating and cooling cycles significantly increased RS content, likely attributed to retrogradation [64]. Starch retrogradation is the process that occurs when chains of gelatinized starch begin to reassociate into ordered structures. In the initial stages, two or more chains linking to each other initiate the formation of increasingly extensive ordered regions. In the cooling process of previously gelatinized starch, low energy levels cause further hydrogen bonding. As the starch chains bond further, the gel structure is tighter, and the water-holding capacity decreases until it reaches crystallinity [66]. Commonly studied storage temperatures for starch retrogradation are 4, 25, or 30 °C or temperature cycling between 4 and 30 °C. It is worth noting that storing starchy products at 4 °C resulted in more rapid crystallization of amylopectin than at 25 or 30 °C [67,68]. In our study, samples were refrigerated for 24 h to ensure microbiological safety and facilitate practical application. The findings demonstrated that the cooling process (24 h, 4 °C) of previously pressurized samples in each product led to a significant increase in the content of RS, as illustrated in Figure 7. This increase occurred irrespective of pressurization time and pressure values and is possibly a consequence of the retrogradation process of starch that underwent gelatinization due to HPP.

4. Conclusions

HPP influences the starch structure, and our study demonstrated that increased pressure and pressurization time impacted the changes in RS content in the examined products. However, changes in RS content are dependent on the tested product and the parameters used. Additionally, the cooling process applied to previously pressurized samples in each product resulted in a significant increase in RS content. This phenomenon is likely attributed to the retrogradation process of starch that has undergone gelatinization due to HPP. All of the tested legumes proved to be rich sources of dietary fiber, especially its insoluble fraction. This study shows the potential of using the HPP method to preserve starchy products based on legumes, but further research on a larger group of products is needed to confirm this effect.