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2023 Volume 2
Article Contents
Abstract
INTRODUCTION
RESULTS
Effects of different seedling ages and disinfection times on seedling germination
Effects of Agrobacterium tumefaciens strains on transformation efficiency
Effects of acetosyringone (AS) concentration in Agrobacterium inoculum on infection efficiency
Effects of Agrobacterium tumefacien concentrations in the inoculum and co-culture time on transformation efficiency
Effects of different concentrations of hormones in co-cultivation medium on callus regeneration
Effects of different concentrations of TMT and Basta on callus regeneration
Effects of hormone concentrations on adventitious shoot elongation
Effects of budding methods on positive plant rooting
Verification of optimization efficiency of watermelon genetic transformation system
DISCUSSION
MATERIALS AND METHODS
Plant materials
A. tumefaciens strains and binary vector construction
Vitro culture and regeneration system
Optimization of the genetic transformation system
Data availability
ACKNOWLEDGMENTS
Conflict of interest
Rights and permissions
References
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REVIEW   Open Access    

Effects of non-starch polysaccharide on starch gelatinization and digestibility: a review

  • 1.

    College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

  • 2.

    Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, China

  • 3.

    College of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, China

More Information
  • Non-starch polysaccharides have been given wide consideration for their use in starch-based food due to their ability to improve texture, sensory attributes, and functional properties of the end product. In a binary system (starch and non-starch polysaccharides), the characteristics of starch, exemplified as gelatinization and digestibility undergo significant changes. This review article, through a combination of origin and chemical structure-based classification approach, explores the impact of non-starch polysaccharides on starch behavior, concretely for gelatinization and hydrolysis. The underlying mechanism to retard gelatinization gives rise to some colloids that can reduce water accessibility and interact with starch molecules, which vary with the origin. The interfering role of starch hydrolysis attributed to polysaccharides restrict starch swelling, the bulk viscosity, and more ordered structures occur in the mixture. Besides, the role of non-starch polysaccharides on enzymes is another factor. Therefore, this paper gives an overview of how non-starch polysaccharides interfere with starch gelatinization and digestion, which provides a comprehensive understanding of starchy products.

  • INTRODUCTION

    The leaf-disc method was initially created to obtain transgenic plants by infecting tobacco leaf explants with Agrobacterium tumefaciens, and the method was simple and effective[1]. However, considering that excellent cultivars or promising application varieties are expected as the initial material, mature and practical transgenic systems are still essential for obtaining more transgenic lines. Watermelon, one crop of the Cucurbitaceae family, plays an irreplaceable role as an economically important fruit crop worldwide. Watermelon has been studied as a model crop for fruit quality due to its unique diversity in color, shape and size, flavor, texture, aroma, and nutrient content[2]. Therefore, personalized watermelon breeding is particularly important.

    Watermelon genetic transformation was first reported in 1994, meanwhile transgenic lines expressing GUS reporter gene were successfully obtained[3]. Thereafter, studies were carried out to improve the regeneration level and transformation rate of watermelon, including genotype, Agrobacterium strains, culture conditions, and chemical agents. Wild watermelon germplasms were more suitable for tissue culture than cultivated watermelon, and shoot regeneration rate were higher[4]. In addition, triploid was superior to diploid in watermelon adventitious bud induction[5]. Various Agrobacterium strains have been applied to watermelon transformation, while there was not explicit exposition regarding the optimum strain[6−10]. Adding acetosyringone (AS) in the Agrobacterium inoculum could improve the sensitivity of strains to watermelon explant wounds and enhance the ability of infection[6, 11]. The infection efficiency and survival rate of explants was the highest after dark cultivation at 23−28 °C for 2−3 d[9,12]. Naphthalene acetic acid (NAA) and 6-benzylaminopurine (6-BA) have been widely used for explants shooting and rooting[7−9, 13, 14], while timentin (TMT), an antibacterial antibiotic, has been applied to many crops, but there was little research on watermelon. In addition, the herbicide glufosinate-ammonium (Basta) was often used as a resistance screening agent in watermelon genetic transformation studies[8, 15].

    When particular genes, such as disease resistance and insect resistance genes, are introduced by means of the transformation system, the transgenic plants keep their original traits, meanwhile, new valuable agronomic traits are added. In addition, approaches for efficient gene-targeting are significant for analyzing functional genes included in the plant genome and producing genetically engineered crops[16]. Previous studies suggested that the CRISPR/Cas9 system[17] was used as a highly efficient and specific tool for accurate genome editing[18]. Because its genome sequence is available[19], the CRISPR/Cas9 system should represent a good prospect for exploring gene functions, improving fruit quality and breeding in watermelon[20]. Transgenic lines with both Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type W (PRSV-W) resistance genes were obtained, and provided great potential for quality breeding of commercial watermelon varieties[7]. Similarly, transgenic lines resistant to Cucumber mosaic virus (CMV) also showed its importance in the watermelon industry[14]. By overexpressing ClTST2, a QTL-mapped gene, the molecular mechanism of sugar accumulation in watermelon fruit was systematically elucidated[8]. In addition, gynoecious watermelon lines obtained by editing ClWIP1, provided a theoretical and technical basis for improving watermelon yield and breeding elite lines[21].

    Prior to this investigation, although watermelon transformation protocols have been described in several studies[13, 14, 21, 22], the efficiency remained as low as 1.67%[9], and research on the optimization of the watermelon transformation system has not yet been reported in detail. An efficient regeneration and transformation system has five characteristics: high efficiency, good reproducibility, simplicity, rapidity and wide applicability[23]. This study attempts to establish an efficient watermelon transformation system and provide a more reasonable way to introduce foreign genes into watermelon.

    RESULTS

    Effects of different seedling ages and disinfection times on seedling germination

    In order to improve the efficiency of seedling germination, gradient tests were carried out for the disinfection time and cultivation time in the dark. The seeds were disinfected with a mixture of 75% alcohol and 6% sodium hypochlorite. The time of disinfection with alcohol was 35 s. The time of disinfection with sodium hypochlorite was designed with three gradients: 8, 12 and 16 min. After sterilization, seeds were rinsed three times with sterile water. The cultivation time of sterilized seeds was carried out in three gradients of 2, 3 and 4 d. Combined with disinfection conditions and dark incubation time, nine different treatments were designed. The seeds were cultured in the seedling culture medium (Supplemental Table S1) at 28 °C under the same dark conditions. The results showed that average germination rates of the seeds in the nine treatment groups (Supplemental Table S2) was between 20% and 100%. There were no significant differences between the germination rates of seeds cultured for 3 d and 4 d when disinfection time was consistent. Combined with the germination states, the highest germination rate can be obtained by sterilizing with 6% sodium hypochlorite for 12 min, and then culturing for 3 d (Supplemental Fig. S1a). The reasons for poor germination rate may be due to the long disinfection time and bacterial lesions resulting from short disinfection time.

    Effects of Agrobacterium tumefaciens strains on transformation efficiency

    According to the results of previous studies, a widely used culture condition was performed in the following experiments[9]. The explants were inoculated in Agrobacterium tumefaciens inoculum at 28 °C, and the initial OD600 of Agrobacterium tumefaciens inoculum was 0.8. After 4 d dark co-cultivation, GFP transient fluorescence was observed. When Agrobacterium strain AGL1 was used for genetic transformation, only weak instantaneous fluorescence was observed (Fig. 1a1, b1). When strain GV3101 was used, medium fluorescence was detected at edges of the whole cotyledon, but stronger fluorescence was found at edges of the whole cotyledon by using strain EHA105 (Fig. 1a3, b3). The results showed that watermelon 'YL' was insensitive to AGL1 strain. Both GV3101 and EHA105 strains could be applicative for the genetic transformation system in watermelon.

    Figure 1.  Transient fluorescence of explants. (a1)−(a3) Bright view. (b1)−(b3) GFP view. (b1) The explants showed weak fluorescence after AGL1 infection. (b2) The explants showed moderate strong fluorescence after GV3101 infection. (b3) The explants showed strong fluorescence after EHA105 infection. Bar = 2 mm.
    DownLoad: Full-Size Img PowerPoint

    Effects of acetosyringone (AS) concentration in Agrobacterium inoculum on infection efficiency

    To determine which concentration of AS in Agrobacterium inoculum could produce the highest genetic transformation efficiency in watermelon. The AS concentrations were separated into five gradients, which were 0, 100, 150, 200, 250 and 300 µM. In each gradient, 120 explants were inoculated. As shown in Table 1, the fluorescence efficiencies of the explants were 45.3%−85.0% due to increased concentrations of AS in the infection solution (Supplemental Table S3). It was also found that the fluorescence efficiency and differentiation efficiency of watermelon explants were significantly different. Fluorescence signals of calli from 100−200 µM AS treatments were acceptable (Fig. 2). However, the infection solution containing 250 or 300 µM AS had large negative effects on germination ability and survival rate of germinated callus, and finally led to a low transformation rate. Our results demonstrated that 200 µM AS in the infection solution was most suitable for genetic transformation of watermelon.

    Table 1.  Fluorescence efficiencies, brightness and differentiation states of watermelon explants under different AS concentrations.
    AS treatment
    (µM)    		Fluorescence efficiency (%)Number of fluorescent explantsFluorescence brightnessCallus differentiation state
    045.3 ± 0.05d54++The calli were green and compact. There was no contamination and little vitrification
    10068.8 ± 0.03b82+++The calli were densely arranged with dark green, fluorescent speckles and little vitrification
    15075.0 ± 0.02b90++++The calli were densely arranged and appeared dark green with bright fluorescence and little vitrification
    20085.0 ± 0.05a102+++++The calli were densely arranged and appeared dark green with bright fluorescence and little vitrification
    25069.3 ± 0.04b83++++Most calli were densely arranged, and a few turned pale and yellowed with water stain
    30054.7 ± 0.05c65++++The calli were closely arranged, partly vitrified with dark green and contaminated by bacteria
    Fluorescence efficiency = Number of fluorescence explants/Total explants number × 100%. Values are means (three independent experiments) ± standard errors (SE), and different letters indicate significant differences between treatments according to Duncan's multiple test (P < 0.05).
     | Show Table
    DownLoad: CSV
    Figure 2.  Fluorescence intensity of the callus under different concentrations of AS. (a1)−(a3) Bright view. (b1)−(b3) GFP view. (a1), (b1) AS concentration at 100 µM. (a2), (b2) AS concentration at 200 µM. (a3), (b3) AS concentration at 300 µM. Bar=1 mm.
    DownLoad: Full-Size Img PowerPoint

    Effects of Agrobacterium tumefacien concentrations in the inoculum and co-culture time on transformation efficiency

    The co-cultivation stage is the key to improving infection efficiency in genetic transformation. During the middle and later periods of co-cultivation, brighter GFP fluorescence meant higher transformation success rate. Due to the browning explants auto-fluorescence, we need to ensure that explants were kept alive and free of pollution as much as possible even though bright fluorescence was found.

    To improve the infection efficiency, four concentrations of A. tumefaciens (EHA105) infection solution were set as 0.6, 0.2, 0.02 and 0.005 at OD600[7, 24]. The co-culture time was set at three gradients of 2, 3 and 4 d. Therefore, 12 associations were listed in total, named A-L respectively (Table 2). Explants, which were cut into eight pieces longitudinally, were immersed in the inoculum for 10 min. The results showed that GFP fluorescence of explants was the strongest when OD600 of A. tumefaciens infection solution was 0.6 and 0.02, but the number of fluorescent explants was more at OD600 = 0.02. The fluorescence efficiency of explants was relatively high when cultured in co-cultivation medium for 3 and 4 d (Fig. 1b). However, when the explants were co-cultured for 4 d, there was pollution around the explants, and the edges showed serious browning. The growth states of each combination are shown in Table 3.

    Table 2.  Transient fluorescence efficiencies and brightness of watermelon explants under different concentrations of Agrobacterium tumefaciens infection solution and co-culture times.
    CombinationAgrobacterium concentration (OD600)Coculture time
    (days)    		Fluorescence efficiency
    (%)    		Fluorescence brightness
    A0.6242.5 ± 1.5b+++
    B0.2254.4 ± 1.4a++
    C0.02238.5 ± 1.8b+
    D0.005233.3 ± 0.3c+
    E0.6338.1 ± 1.1c+
    F0.2343.0 ± 2.0bc++
    G0.02379.1 ± 0.9a+++
    H0.005347.1 ± 0.5b+
    I0.6435.5 ± 1.4c+
    J0.2444.5 ± 2.6b+
    K0.02479.1 ± 0.6a+++
    L0.005451.5 ± 2.5b++
    Fluorescence efficiency = Number of fluorescence explants/Total explants number × 100%. Values are means (three independent experiments) ± standard errors (SE), and different letters indicate significant differences between treatments according to Duncan's multiple test (P < 0.05).
     | Show Table
    DownLoad: CSV
    Table 3.  Growth states of watermelon explants under different Agrobacterium tumefaciens infection solution and co-culture times.
    CombinationGrowth state
    AA small part died, and the margin of the surviving explants were yellow with obvious water-stained flora
    BThe edges showed traces of a watery microflora
    CThe explants were well developed
    DThe explants were well developed
    ESome died with serious spillage, and fluorescence
    explants were browning
    FA small portion were dead and the margins browned
    GThe explants dilated well
    HThe explants dilated well
    IMost died with serious spillage phenomenon, and
    virtually all the edges browned
    JPartially dead with serious spillage and serious edge browning
    KPartially edged with yellow and with spillage phenomenon
    LThe explants were well developed
     | Show Table
    DownLoad: CSV

    Effects of different concentrations of hormones in co-cultivation medium on callus regeneration

    The direct differentiation method was used to optimize genetic transformation of watermelon, while the calli redifferentiation method was an optimization system developed on the basis of previous experience in this research[25]. According to the results of seedling germination, experiments of cotyledon dedifferentiation were conducted. The strong calli were more easily induced into adventitious shoots by 6-BA, which was beneficial to improve the efficiency of genetic transformation efficiency. The temperatures of cotyledon differentiation ranged from 26−28 °C. Concentration treatments of hormones were divided into 12 groups (Supplemental Table S4). We further selected 3 and 4 d (named as YL-3 and YL-4 in Supplemental Table S4) to culture explants in co-cultivation medium (Supplemental Table S5). The results indicated that the combination of 1.5 mg/L 6-BA without indole acetic acid (IAA) had the highest callus differentiation rate after 3 d of co-cultivation.

    Effects of different concentrations of TMT and Basta on callus regeneration

    Ticarcillin, one of the active components in TMT, was used as an antibacterial agent. Based on the differentiation and growth state of callus, TMT with a concentration of 50−350 mg/L could inhibit the growth of bacteria at an early stage. However, concentrations of TMT at 350 mg/L caused more severe aetiolation in the later stages of development, especially in the rooting stage, which could affect the growth of the roots (Supplemental Table S6). The addition of TMT with a concentration at 200 mg/L had no fungal contamination. Based on the standard of high proliferation rate and normal growth state, 200 mg/L TMT was considered as the best concentration, which was added to the recovery medium (Supplemental Table S7). In addition, 200 mg/L TMT could effectively inhibit the growth of Agrobacterium tumefaciens and no contamination was detected after regenerated explants were transferred to a TMT-free culture medium. TMT with a concentration higher than 350 mg/L resulted in yellowing phenomenon and seedling death, which indicates that excessive TMT has some toxic effect. We named the recovery stage after the recovery process, as shown in the Supplemental Fig. S1c.

    The gene editing vector used in this experiment contained a marker gene that could be resistant to Basta, and resistance screening was used to screen the positive transgenic shoot (Supplemental Table S8). At concentrations of 0.4 and 1.4 mg/L Basta, the survival rate and fluorescence efficiency of callus were higher than those of other concentration gradients, although there was no obvious difference between them (Table 4). However, when the concentration of Basta was 2.4 mg/L, the callus vitrified seriously, and the redifferentiation of calls were inhibited. At 1.4 mg/L, the well-grown callus showed high fluorescence efficiency, and the brown callus was selected as the best concentration. We named the selection stage after the selection process (Supplemental Fig. S1d). The results revealed that the concentration of Basta higher than 1.4 mg/L caused the shorter survival time of explants.

    Table 4.  The survival rates and growth states of callus under different concentrations of Basta.
    Basta concentration
    (mg/L)    		Callus survival rate
    (%)    		Fluorescence rate of
    survival callus (%)    		Callus state
    0.4100.0 ± 0.0a69.1 ± 4.1cThe tissues appeared dark green and grew well
    1.473.6 ± 0.4b78.7 ± 0.3bPartial tissues appeared brown and most grew well
    2.473.0 ± 3.0b80.5 ± 1.5bPartial tissues were transparent and light green with serious vitrification
    3.440.8 ± 1.2c92.0 ± 2.9aPartial tissues died with serious yellowing phenomenon and obvious spillage
    4.40.7 ± 0.3d0.7 ± 0.3dMost tissues died, and the spillage was obvious
    Callus survival rate = Survival callus number/Total callus number × 100%; Fluorescence rate of survival callus = Survival callus number with fluorescence/Total survival callus number. Values are means (three independent experiments) ± standard errors (SE), and different letters indicate significant differences between treatments according to Duncan's multiple test (P < 0.05).
     | Show Table
    DownLoad: CSV

    Effects of hormone concentrations on adventitious shoot elongation

    Adventitious shoot differentiation was easier in well-developed and dedifferentiated calli. In order to improve the regeneration rates of buds, three concentrations of 6-BA (0.05, 0.1, 0.15 mg/L) and two concentrations of naphthlcetic acid (NAA) (0.05, 0.1 mg/L) were applied to analyze the formation of adventitious elongated shoots (Table 5). When the concentration of 6-BA is 0.1 mg/L and NAA is 0.1 mg/L, the frequencies of shoot elongation was the highest (Fig. 3a). When 6-BA and NAA were higher than 0.15 mg/L and 0.1 mg/L respectively, the callus directly differentiated into aerial roots, and explants were prone to yellowing (Fig. 3b). When 6-BA and NAA were 0.1 mg/L and 0.05 mg/L respectively, shoot elongation was inhibited (Fig. 3c). The results indicated that 0.1 mg/L 6-BA and 0.1 mg/L NAA were the best concentrations to improve the elongation efficiency of adventitious shoots and keep their good growth states (Supplemental Fig. S1e). Shoot elongation medium is shown in Supplemental Table S9.

    Table 5.  Adventitious shoot elongation efficiencies and shoot growth states under different concentrations of hormones.
    6-BA (mg/L)NAA (mg/L)Shoot regeneration
    rate (%)    		Adventitious shoot growth state
    0.050.0510.3 ± 1.8cThe adventitious shoots differentiated less and had serious vitrification
    0.10.0522.0 ± 1.0bLess differentiation, and vitrification was not serious
    0.150.0532.4 ± 2.6aLess differentiation, some shoots unable to continue to elongate and showed yellowing
    0.050.129.2 ± 0.7bLess differentiation, calli partly yellowed and vitrified
    0.10.164.5 ± 5.5aWith more differentiation, calli partly vitrified without yellowing phenomenon
    0.150.145.6 ± 3.8bMore differentiation, shoots partly yellowed with a small amount producing air roots
    Shoot regeneration rate = Number of regerminated shoots/Total explants number × 100%. Values are means (three independent experiments) ± standard errors (SE), and different letters indicate significant differences between treatments according to Duncan's multiple test (P < 0.05).
     | Show Table
    DownLoad: CSV
    Figure 3.  Redifferentiation state of explants under different hormone concentrations. (a) The callus differentiated well under 0.1 mg/L concentration of 6-BA and 0.1 mg/L concentration of NAA. (b) The callus redifferentiated to form aerial roots under 0.15 mg/L concentration of 6-BA and 0.1 mg/L concentration of NAA. (c) The callus appeared to yellow and could not differentiate under 0.15 mg/L concentration of 6-BA and 0.05 mg/L concentration of NAA. Bar = 12 mm.
    DownLoad: Full-Size Img PowerPoint

    Effects of budding methods on positive plant rooting

    In this study, it was found that adventitious buds could generate roots 7−14 d after being transferred to the rooting medium. (Supplemental Table S10). Based on these results, all optimized aspects were used in our following research to obtain an effective transformation frequency. A positive plant was obtained, which is shown in Supplemental Fig. S1f.

    Verification of optimization efficiency of watermelon genetic transformation system

    The above experimental results showed that the watermelon transformation system had been optimized to some extent. To verify the efficiency, we selected ClREC8 (Cla97C07G132920), ClACS1 (Cla97C01G017090) and ClACS7 (Cla97C03G066110) as target genes to perform gene knockout assay in watermelon. PCR amplification was performed using the primers Cas9-F and Cas9-R, and the results revealed the transgenic plants contained exogenous T-DNA inserts (Fig. 4). Furthermore, targeted gene sequences in the transgenic plants were sequenced, and the results in three target genes were partly shown (Fig. 5, Supplemental Fig. S2). As a result, in approximately 300 inoculated explants, we obtained a total of 45 T0 transgenic lines, among which 42 plants were successfully edited. The percentage of 93.3% meant that the optimized transformation system could also be used for watermelon gene editing.

    Figure 4.  PCR analysis of the transgenic plants. Genomic DNA isolated from putative transgenic plants were subjected to PCR amplification with Cas9 primers. Lane M, Trans2K Plus DNA Marker; Lane P, positive control (plasmid); Lanes 1, 2, 3, 5, 7, 8, 12, 13, 14, 15, 17, 19 and 20, putative transgenic watermelons; Lane 4, 6, 9, 10, 11, 16, 18, 21 and 22, Non-transgenic watermelon.
    DownLoad: Full-Size Img PowerPoint
    Figure 5.  Targeted mutagenesis of (a) ClREC8, (b) ClACS1, and (c) ClACS7 in the transgenic lines. The schematic diagrams illustrate sgRNA targeting the exons. The target sequences are shown in orange with protospacer adjacent motifs (PAM) sequence highlighted with black rectangles. Nucleotide deletions are shown with blue dashes, and inserted nucleotides are shown in green.
    DownLoad: Full-Size Img PowerPoint

    DISCUSSION

    It is widely known that transgenesis and gene-editing technologies were made full use of for promoting plant research processes [25−27]. Considering the significance of the genetic stability of both transformed exogenous genes and regenerated plants, various methods have been tried for transformation, including particle acceleration[28], electroporation and polyethylene glycol permeabilization of protoplasts[29], and DNA transfer mediated by Agrobacterium. However, previous studies indicated that only few positive lines have been obtained due to difficulties in watermelon transformation[17, 30]. Extremely low transformation efficiency in watermelon transformants was largely due to the high degree of escape and chimeric shoot[14].

    In this study, we optimized the watermelon genetic transformation system to improve the genetic transformation efficiency. The growth process of young seedlings grew promisingly compared with old seedlings[31], however, both tender aged and over aged seedlings are not suitable for better yield[32]. The watermelon seedling age, which was of great significance for callus regeneration, was the key factor as to whether induction was successful and highly effective or not. The explants state determined the impregnation efficiency of the subsequent Agrobacterium-mediated method. Poor explants state usually causes problems such as bacterial growth, vitrification or redifferentiating inability in the cultivation process. The experimental results showed that explants with good viability could be obtained after a disinfection time of 12 min and cultivation time of 3 d.

    The findings of this study were consistent with other reports of watermelon transformation with the Agrobacterium-mediated method[3]. AS, a phenolic compound which was a known vir gene inducer especially in monocotyledon plants, plays a crucial role in the transferring process[33]. However, a high quantity of AS was toxic to the plants, which could be mainly due to the presence of alcohol as a solvent[34]. Germplasm differences showed different sensitivity to AS. In many other crops, such as wheat, cucumber and sesbania, the addition of AS to Agrobacterium inoculum and co-cultivation medium has been shown to improve conversion efficiency[35−37]. Previous studies indicated that the highest concentration of AS used for transformation of various plants was between 100−200 µM[38−40]. We selected an AS concentration at 200 µM to improve infection efficiencies of strain and fluorescence efficiency.

    Due to germplasm differences possessing diverse genotypes, efficiencies of watermelon genetic transformation were also different[41]. It was also essential to select the appropriate Agrobacterium strain for different watermelon germplasms to improve their ability in infecting explants. In previous studies, types of Agrobacterium strains used in the genetic transformation of cucurbitaceae crops were different, including AGL1, EHA105 and GV3101[6, 42]. Our study indicated that watermelon germplasm 'YL' was sensitive to Agrobacterium strain EHA105, which was the basis for improving the transformation efficiency.

    Agrobacterium concentration and coculture time were considered as the key to improving the genetic transformation efficiency. Too low a c oncentration could not infect explants smoothly, whereas too high a concentration led to bacterial overgrowth, causing explants death. Too short coculture time could not ensure T-DNA transferred into the explant cells, and integrated into the genome. Instead, bacterial overgrowth resulted in explants not being able to continue to grow, and transformation efficiency was reduced. In terms of Agrobacterium population density, the fluorescence efficiency of watermelon explants was higher at an OD600 of 0.02. The total time was fixed at 10 min, which was the same time reported previously for immersion in Agrobacterium inoculum[43]. The optimum working concentration of Agrobacterium used in this study was relatively low, and the fluorescence was brighter when co-cultured for 3 d. This may be related to the sensitivity of explants from different germplasms. However, if infected by lower concentrations, the efficiency cannot be improved. In contrast, co-cultivation for too long would cause bacteria colonies proliferation and explants death.

    Efficiencies of watermelon transformation were affected by many other factors, and certain parameters were slightly different in a variety of watermelon germplasms[44]. Taking watermelon germplasm ‘YL’ as the original material, this study further explored hormone concentrations for watermelon callus development. The explants differentiation rate was the highest with 1.5 mg/L 6-BA and no IAA. In addition, adventitious shoots could be induced to differentiate and even elongate by using the hormone combination of 0.1 mg/L 6-BA and 0.1 mg/L NAA, and growth states were good. The results indicated that ‘YL’ might not be sensitive to IAA.

    Antibiotics could effectively inhibit a portion of Agrobacterium to ensure good growth of explants. TMT, an antibiotic agent that is a combination of ticarcillin and clavulanic acid, can positively replace other antibiotics like carbenicillin and cefotaxime in tissue culture[45, 46]. Because it is stable in solid agar medium and can keep effective for longer than 70 d, TMT could be an alternative antibiotic for suppressing Agrobacterium growth in transformation effectively and improving regeneration potential compared with other antibiotics such as carbenicillin[47, 48]. Low concentration of TMT could not arrest bacteria proliferation so that explants cannot continue to grow. In contrast, high concentrations of TMT would not only inhibit the bacterial growth but also inhibit the growth of explants, resulting in a decline in transformation efficiency. Our results demonstrated that the most appropriate concentration of TMT was 200 mg/L, which could facilitate shoot regeneration.

    A codon-modified phosphinothricin acetyltransferase gene, which confers resistance to the herbicide glufosinate-ammonium, was used as the selectable marker[49]. Glufosinate-ammonium is able to block the activity of an enzyme used in the biosynthesis of amino acid glutamine, which has been reported in different species[50−52]. The study demonstrated that Basta had a pivotal effect on the screening of positive callus and shoot at 1.4 mg/L. The screening method could greatly improve the detection efficiency, and reduce the workload.

    The experiments provided preparation for the subsequent obtaining of knocked lines and reduced the problems such as poor differentiation ability of callus, yellowing phenomenon, and vitrification in watermelon. Meanwhile, fluorescence efficiencies of the watermelon callus, which was conducive to the fluorescent shoots, were improved. The pH and humidity of each medium should be strictly controlled in the experiment, and the callus growth states should be observed regularly and repeatedly.

    Recently, a study revealed that a mutation in the miR396 microRNA region included in ClGRF4 gene led to efficiency up to 67.27% in watermelon, no matter which genotype was applied for transformation[53]. Nevertheless, our results suggested that watermelon transformation efficiency (from explant to transgenic plant) was increased to 12.5% using the optimized system in this study. Multigene knockouts as well as gene replacements have not yet been widely studied in watermelon and other plants[54]. Our research made it possible to optimize the watermelon multi-genetic transformation system more efficiently in the future.

    MATERIALS AND METHODS

    Plant materials

    The watermelon germplasm 'YL', cultivated at 36o−39o N and 107o−111o E from a warm semi-humid climate, has strong drought resistance and moderate resistance to Fusarium wilt, and was used as the original material in this experiment. The materials were planted in a greenhouse at the College of Horticulture, Northwest A&F University (Shaanxi, China).

    A. tumefaciens strains and binary vector construction

    Three A. tumefaciens strains including AGL1[55], GV3101[6] and EHA105[56] were tested in this study. The binary CRIPSR/Cas9 vectors pBSE402 (carrying a Bar and a GFP genes, both of which were driven by 35S promoters) was modified from pBSE401 provided by Dr. Qijun Chen from China Agricultural University (Beijing, China), and the vectors were constructed as described[7,16]. Positive regenerated plants were detected by polymerase chain reaction. The PCR primers Cas9-F (5’-GCAGCTCTCCAAGGACACAT-3’) and Cas9-R: (5’-CGTGAGTTCTTCTGGCCCTT-3’) were designed using Primer Premier 5 software. The PCR was conducted using Taq PCR Mix (GenStar, China), and performed in an optical 96-well plate with 2720 Thermal Cycler (Applied Biosystems, USA). The positive results were further sub-cloned with pUC18 plasmids, sequenced, and analyzed according to Kaur et al.[57].

    Vitro culture and regeneration system

    Plants were regenerated from watermelon cotyledons[58, 59]. Adventitious shoots formed on the proximal cut edges of the cotyledonary explants[60]. Cotyledons of sterile watermelon seedlings aged 2−5 d were cut into small pieces of 0.5 cm, then inoculated on MS medium with 6-BA to dedifferentiate into calli, on which a lot of adventitious shoots appeared after 2 weeks. Adventitious shoots were then transferred to the rooting medium for 2−3 weeks. The culture conditions were 28 °C, 16 h day, and 3,000 lx light intensity.

    Optimization of the genetic transformation system

    The experiment was carried out from several aspects to optimize the transformation system, including different seedling ages, Agrobacterium strains, AS and Agrobacterium concentrations of the inoculum, concentrations of antibiotic TMT and Basta selection pressure, and concentrations of added hormones.

    Data availability

    The data that support the results are included in this manuscript and its supporting information files. Other relevant materials are available from the corresponding author upon reasonable request.

    ACKNOWLEDGMENTS

    This work was supported by Young Talent fund of University Association for Science and Technology in Shaanxi, China (20210202, to JS), and the Science and Technology Innovation Team of Shaanxi (2021TD-32, to ZL).

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Li S, Chen W, Zongo AWS, Chen Y, Liang H, et al. 2023. Effects of non-starch polysaccharide on starch gelatinization and digestibility: a review. Food Innovation and Advances 2(4):302−312 doi: 10.48130/FIA-2023-0029
    Li S, Chen W, Zongo AWS, Chen Y, Liang H, et al. 2023. Effects of non-starch polysaccharide on starch gelatinization and digestibility: a review. Food Innovation and Advances 2(4):302−312 doi: 10.48130/FIA-2023-0029
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Effects of non-starch polysaccharide on starch gelatinization and digestibility: a review

  • 1.

    College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

  • 2.

    Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan 430070, China

  • 3.

    College of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, China

  • Received: 20 July 2023
  • Accepted: 10 October 2023
  • Published online: 08 December 2023
Food Innovation and Advances  2,  2023, 2(4): 302−312  |  Cite this article

Abstract: Non-starch polysaccharides have been given wide consideration for their use in starch-based food due to their ability to improve texture, sensory attributes, and functional properties of the end product. In a binary system (starch and non-starch polysaccharides), the characteristics of starch, exemplified as gelatinization and digestibility undergo significant changes. This review article, through a combination of origin and chemical structure-based classification approach, explores the impact of non-starch polysaccharides on starch behavior, concretely for gelatinization and hydrolysis. The underlying mechanism to retard gelatinization gives rise to some colloids that can reduce water accessibility and interact with starch molecules, which vary with the origin. The interfering role of starch hydrolysis attributed to polysaccharides restrict starch swelling, the bulk viscosity, and more ordered structures occur in the mixture. Besides, the role of non-starch polysaccharides on enzymes is another factor. Therefore, this paper gives an overview of how non-starch polysaccharides interfere with starch gelatinization and digestion, which provides a comprehensive understanding of starchy products.

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    Introduction

    • Starch plays a considerable role in offering energy to humans mainly in digestible carbohydrate form, and is exerting numerous uses in versatile food recipes[1]. However, relying solely on starch-based foods may not satisfy the diverse needs of consumers. Moreover, the intake of foods that are predominantly composed of rapid digestion starch causes a fluctuation in blood glucose, and in the long term a decrease in insulin sensitivity, which is not suitable for individuals with certain diet-related chronic diseases[2]. To address these challenges, non-starch polysaccharides (NSPs) are incorporated into starch-based foods. NSPs refer to the nonstructural complex polysaccharides joined through glycosidic linkages, in addition to starch, which consists of numerous monosaccharide units. Given their safety, good biocompatibility, and biodegradability, NSPs have been utilized for stabilizing, rheological improvement, and emulsifying in the food industry[3]. When combined with starch, NSPs can modify the basic properties of starch, which improves the benefits of the mixture system.

      Gelatinization is an instrumental procedure in the fabrication of starchy foods, involving the swelling process, amylose dissolution, birefringence disappearance, and transformation from ordered structure to disordered[4]. When the binary system is formed (non-starch polysaccharide with starch), the gelatinization process of starch will be undoubtfully affected. Many reports have reported the role of NSPs in interfering with starch gelatinization[5,6]. NSPs can cause variations in starch gelation temperature and changes in endothermic enthalpy. The difference depends highly on the NSPs' characteristics, the origin of starch, and their interaction manner. Most hydrocolloids exert an adverse role on the starch gelatinization profile, resulting in incomplete gelatinization or hindered process, which ultimately affects its functional properties such as digestibility.

      In terms of the digestion properties, the influence of NSPs on gelatinization directly relates to the breakdown and absorption of starch in food. During digestion, starch is degraded into oligosaccharides by amylase, which is then absorbed into the bloodstream through the villi of the small intestine. Various NSPs play a role in modulating starch hydrolysis, resulting in reducing rapidly digestible starch (RDS) and accordingly raising the content of slow and resistant starch[7]. However, variations were encountered with starch and hydrocolloids from various origins. Thus, NSPs' addition resulted in a different distribution between starch fractions. Researchers have explored the underlying mechanism by which NSPs affect starch digestibility. Most share the opinion that viscosity plays a critical role[8,9] as it can retard starch swelling, hinder the collapse of the starch structure, and induce different changes in the crystalline regions based on the source and type of colloids[10]. However, it is important to note that the viscosity of hydrocolloids alone does not solely determine starch hydrolysis, as the interaction between the composition of the colloid and starch or enzymes also plays a crucial role[11].

      This paper aims to present a comprehensive summarization of the effect of NSPs on starch gelatinization and digestion properties. The NSPs covered different origins and functions. The underlying mechanisms of NSPs played on starch gelatinization and hydrolysis were also discussed. Therefore, this manuscript provided an overview of the NSP's role in starch properties to promote healthy starchy-based food development.

    Effect of NSPs on starch gelatinization

      Starch gelatinization

    • Gelatinization is a crucial step occurring in many starchy food operations, for example, the extrusion of cereal-based products and baking, etc. A profound understanding of starch in terms of the fundamental molecular interactions of the gelatinization process is vital for its industrial applications[12].

      Gelatinization gives rise to the irreversible changes occurring in the starch structure associated with loss of birefringence, starch granules swelling, amylose leaching, and viscosity changes[13]. It is a molecular transition process with the underlying mechanism described as the amorphous growth rings once contact with excess water molecular will swell firstly (breaking of hydrogen bonds), and then the semi-crystalline lamellae change accordingly, which leads to the decrease of crystallinity of the granule[14,15]. Slade & Levine believe that the dissolution of amylose leads to increasing viscosity, which is not before the amorphous region's glass transition or melting is completed[16]. Moreover, Waigh et al. suggest that the gelatinization process involves two steps in high water circumstances. The slow dissociation can take the place of the helix-helix in molecular evidenced by the crystalline smectic-nematic test parameters first. The second stage is related to the transition from helix to coil which is accompanied by the helices unwinding[17].

      In fact, during food industry utilization, starch is not merely a component, and kinds of hydrocolloids exist to overcome the gap between research and application. Moreover, the influences of NSPs on starch gelatinization appear to vary due to multiple factors. For example, the native characteristic of polysaccharides such as their origin, structures, molecular weight ranges, ionic charge, and flexibility can influence their role. Plenty of research focused on the gum on starch gelatinization[18]. In this review, we examine the effects of NSPs from different origins, based in part on the classification proposed by Kumar et al.[19], which is mainly categorized based on chemical structures. Moreover, this paper not only focused on botanic-origin non-starch polysaccharides but NSPs derived from animal and microbial origin were also included.

      NSPs

    • NSPs are supposed to be the nonstructural complex polysaccharides except for starch, which is made up of various monosaccharide units, which mainly form on the linkage through β-glycosidic bonds (Table 1)[19]. In this review, the term NSPs refer to gums, which are generally considered safe for human consumption, and widely used for versatile functionalities such as thickening, gelling, stabilizing, or emulsification[20]. Based on the reaction with water, NSPs can be characterized into two groups. Soluble NSPs such as pectin, inulin, konjac glucomannan, and β-glucan, often increase viscosity. While insoluble NSPs can serve as water-binding reagents for their fecal-bulking capacity[21]. Though many kinds of criteria are used to classify the term of NSPs, based on Bailey's recommendations, the most preferred classification method was chosen to organize NSPs here to avoid ambiguity, and at the same time take into account chemical structure[22]. Firstly, we classified the NSPs into three categories (according to the origins), in terms of the botanical category (namely plant cell wall structural polysaccharide), the NSPs are divided into three sections, namely the cellulose and non-cellulosic polymers and pectic substances, according to their function in the cell wall. Cellulose is the fiber polysaccharide in the cell wall which acts as the fiber microfibrils, non-cellulosic polymers function as fiber matrix and pectic polysaccharides serve as the intercellular cement. Lastly, based on different chemical structures, the non-cellulosic polymers fall into two main groups (pentosans and hexosans that are pentose-free). Figure 1 outlines the detailed classification scheme.

      Table 1.  Summary of important molecular characteristics of some common non-starch polysaccharides used in foods.

      OriginNameSolubilityMajor compositionMolecular weight (kDa)Main functionReference
      Botanical
      Cellulose derived moleculesMethyl CelluloseSolubleβ (1,4) D-glucose20~1,000Thickening, gelling, stabilizing, emulsification[71,73]
      Cellulose derived moleculesCarboxy methylcelluloseSolubleβ (1,4) D-glucose95~1,100Thickening[74]
      Cellulose derived moleculesHydroxypropyl methylcelluloseSolubleβ (1,4) D-glucose20~1,000Thickening, gelling, stabilizing, emulsification[74]
      Plant tissue extractsPectinSolubleα-(1–4)-linked D-galacturonic and mannuronic acid.50~150Stabilizing, gelling[71,73]
      Tree gum exudates (Acacia Sap)Gum ArabicSolubleGalactose200~800Emulsification, film forming[71,73]
      Roots of chicory (Asteraceae)InulinSolubleβ-D-fructose0.5~13Prebiotic, thickening[71]
      TubersKonjac-glucomannanSolubleD-glucose and D-mannose,10~2,000Thickening, gelling, texturing, water binding[73]
      Viscous plant substances (Seeds mucilages)Locust bean gumSolubleD-mannose and D-galactose500~1,000Stabilizing, thickening,[71,73]
      Viscous plant substances (Seeds mucilages)Tara gumSolubleD-mannose and D-galactose~1,000Stabilizing, thickening, gelling[71]
      Plant tissue extractsβ-glucanSolubleD-glucose10~1,000Stabilizing, thickening, emulsification[74]
      Seed endosperm of Cyamopsis tetragonolobusGuar gumSolubleLinear chain of Galactomannan unit100~2,000Stabilizing, thickening[73]
      Tree gum exudates (Dried sap of several legumes of the Astragalus, including A.
      adscendens, A. gummifer,
      and A. tragacanthus)      		Tragacanth gumSoluble: tragacanthin; Insoluble: bassorinTragacanthin and tragacanthic acid~840Stabilizing, thickening, emulsification[73,74]
      Viscous plant substances (mucilages)PsylliumSolubleArabinoxylan35~3,800Thickening, gelling[74]
      Brown seaweedsAlginateSolubleβ-D-Mannuronic Acid32~400Stabilizing, gelling[71]
      Red seaweeds (Sphaerococcus euchema)AgarSoluble in hot waterβ-D-Galactopyranose80~140Stabilizing, gelling[71,73]
      Red seaweedsCarrageenan (kappa-, lambda- and iota-)SolubleSulphated D-galactose and L-anhydrogalactose400~700Stabilizing, gelling, thickening[71,73,75]
      Animal
      Crustaceans, InvertebratesChitosanSoluble in acetic aqueous solutions2-amino-2-deoxy-β-D-glucose4~500Gelling[73,77]
      Microbial
      Aureobasidium
      pullulans      		PullulanSolubleα-D-glucan40~600Thickening, gelling, foaming, flocculating, stabilizing, binding[10,75]
      Fermentation gums (Xanthomonas campestris exudate)Xanthan GumSolubleβ-D-glucose u, two mannose and one glucuronic acid1,000~50,000Structure formation, thickening, stabilizing[71,73]
      Fermentation gums (Pseudomonas elodea)Gellan gumSoluble in hot waterThe basic unit is composed of 1,3- and 1,4-linked 2 glucose residues, 1,3-linked
      1 glucuronic acid
      residue, and 1,4-linked
      1 rhamnose residue      		~500Gelling, film forming[74−76]
      Fermentation gums
      (of microbial origin)      		CurdlanSoluble in an alkaline aqueous solutionlinear glucan D-glucose53~5,800Gelling[77]
      Fermentation gums
      (of microbial origin)      		DextranSolubleComposed of D-glucose, the main chain is α-1,6 bonds, and there are also branched chains with
      α-1,4 or α-1,3 bonds      		40~70Stabilizing, thickening, emulsification[74]

      Figure 1. 

      The classification scheme of non-starch polysaccharides (NSPs). Italics represent the NSPs chosen under each category to depict the effect on starch. Cellulose serves as the fiber microfibrils, non-cellulosic polymers serve as cell walls or fiber matrix, and pectic substances function as intercellular cement.

    NSPs of botanic origin

      Cellulose derivatives

    • Carboxymethyl cellulose (CMC) is a homopolysaccharide that has been extensively used in food research and industry. Zhou et al. reported that CMC with the wheat starch mixture was accompanied by a higher To and Tc and endothermic enthalpy, which may be the result of the association of NSPs with starch, thus changing the mobility of the starch chain[23]. Nixtamalization maize dough with CMC was made by Andres et al., whose research suggests similar results, namely, the addition of CMC brought about a surge of thermal parameters of maize starch though the gelatinization enthalpies values decreased when the NSPs concentration increased[24].

      Non-cellulose categories

      β-glucans

    • β-glucans as a category of non-starch polysaccharides that can be obtained from many cereals, such as oats, barley, and wheat, mainly through the β-glycosidic linkage in different ratios of β-1,3 and β-1,4. The β-glucans, which have various chemical structures, can serve as gelling and stability agents in food recipes[25]. Satrapai & Suphantharika stated that the thermal properties of mixtures (rice starch/β-glucan ) switch to a higher level, while ΔH declined with the increasing amount of NSPs[26]. This may be explained as limiting water mobility. While Rawiwan & Suphantharika found nearly no effect of β-glucans on rice starch[27].

      Inulin

    • Luo et al. estimated three kinds of inulin on wheat starch thermal properties[28]. As inulin increased, there was a slight increasing trend in terms of To, and the effect may be more evident when the additives are at higher levels due to the hydration of NSPs. Peak temperatures (Tp) increased with the addition of concentrations of inulin, while Te varied depending on the degree of polymerization. As inulin has a lower degree of polymerization (DP), it plays a more significant role in ΔH because the smaller polysaccharide could easily interfere with the orderly assembled crystallized region and double-helical architecture.

      Arabinoxylans

    • Arabinoxylans' effectiveness in starch gelatinization keens on the molecular weights[29]. Low molecular weight, water-extractable arabinoxylan plays a more evident role in the inhibition of amylose leaching. Corn fiber gum exhibits a similar influence on the maize starch, accompanied by the concentration increase. It can interact with the amylose molecules, which would hinder starch granule breakdown[5]. This interaction occurs through entanglements and hydrogen bonds, thus stabilizing the system[30].

      Tamarind

    • Tamarind seed polysaccharide can increase three kinds of corn starch thermal transition temperature (Tp), which shows a negative effect on starch gelatinization. This effect is attributed to the binding capacity between tamarind seed polysaccharide and starch granules and changes that occur in the molecular conformation of starch[31]. Consequently, the starch/non-starch polysaccharide systems exhibit higher ΔH.

      Fenugreek gum

    • Fenugreek gum lifted the onset temperature of viscosity and a reverse trend was observed when the starch concentration was lower[32]. Moreover, when the concentration of starch is higher (15%), the endothermic enthalpy value remains unchanged[33]. The discrepancy is because of the larger volume effect at higher concentrations on the rheological properties than the molecular associations.

      Konjac glucomannan

    • Konjac glucomannan (KGM) brings a surge in parameters (To, Tp, Tc) with no changeable enthalpy[34]. Schwartz et al.[35] reported the effect on potato starch depends highly on the KGM concentration and water content. To was unchanged and Tc increased as more KGM occurred. It is often assumed that the enthalpy decreases with the increase of KGM and declined water content, which is mainly caused by the unable fully gelatinization when limited water exists[35].

      Guar gum

    • Guar gum has been frequently investigated by researchers in the past years in case of interfering with starch gelatinization. Torres et al. suggested guar gum reduces the availability of water, which owing to its hydrophilic nature, leads to lower starch hydration and consequently lower associated enthalpy when the gum concentration is 0.5%. Guar gum delays chestnut starch gelatinization[36]. The parameters related to the second peak both shifted higher with the increasing guar gum. Moreover, guar gum can also limit granule swelling and constrain amylose leaching[37]. In terms of acorn starch, guar gum retard the gelatinization and decreases the ΔH[38], which gives rise to the reduction in the hydration capacity of the mixture systems[39]. Though some exceptions were detected, as Mali et al. reported, the guar gum had a negligible effect on yam starch either transition temperature or enthalpy[40].

      Carrageenan and Alginate

    • NSPs derived from algae, such as carrageenan and alginate, are commonly used as polyhydroxy compounds to enhance the properties of starch slurries. This approach is considered safe and effective, offering advantages over chemical modification and enzymatic hydrolysis methods. Sodium alginate and stearic acid can raise the starch onset temperature, which suggests the hydrocolloid would delay the gelatinization process while decreasing the enthalpy by 5.7−6.7 J/g[41]. Carrageenan, on the other hand, protects starch granules and contributes to achieving the desired texture for the starch-based formulation. Carrageenan shows different impacts on the aqueous starch gelatinization profile, mainly because the thermodynamic incompatibility of the polysaccharide with the amylose and phase arrangements occurs[42].

      Pectin substances categories

      Pectin polysaccharides

    • Pectin polysaccharides can be classified into high and low-methoxylated kinds, based on the degree of esterification[43], which makes their difference in properties. However, it seems that both high and low methoxylated pectin can raise the temperature of cornstarch gelatinization and decrease the ΔH[44]. It seems that the concentration of pectin is more important than variety. When a higher level of pectin exists, the transition temperature shifts to a higher trend, especially for potato starch. In contrast, inulin has a different tendency, which depends on its DP. As reported by Teresa et al. the medium DP inulin exerted a prominent role in interfering with potato starch gelatinization and the inferior role played by the lowest DP[45].

      Extracts or mucilage

    • Arranz-Martínez et al. did not find the effect of NSPs in both waxy rice and non-waxy rice starch, as well as the enthalpy[9]. The same result was conveyed by Liu et al. who found the yellow mustard mucilage had no inferring effect on wheat or rice starch gelatinization temperature only causing a slight increase in melting enthalpy[46]. However, Alamri et al. studied the okra extract with starch blends. The NSPs namely okra extract retards the starch gelatinization by raising the peak temperature. Moreover, the okra extract can perform an indirect role through interaction with water molecules[47]. In terms of Mesona chinensis polysaccharide, it relies heavily on structure associated with extraction methodology when interacting with starch[48].

    NSPs of animal origin

      Chitosan

    • When it comes to polysaccharides of animal origin, chitosan serves as the most representative example. In acidic media, chitosan acts as a cationic polysaccharide. When comparing the starch thermal properties in the presence of positively charged polysaccharides, researchers found that chitosan can increase the DSC onset gelatinization temperature and show more effectiveness in terms of the lab-made maize starch than the commercial one[49]. Different results have been given where researchers suggest that the effect depends on the amount of polysaccharide. Specifically, when the chitosan level is below 5%, there seems to be no significant effect[50]. Interaction between starch and polysaccharide solution was stronger, giving rise to increasing gelatinization parameters, conversely, when starch interacts with water molecules predominately, the effect will tend to reverse[51].

    NSPs of microbial origin

      Xanthan

    • Viturawong et al. reported that xanthan did not modify the rice starch thermal parameters besides the enthalpies were significantly decreased[52]. The effects were more pronounced when there was xanthan gum with higher molecular weight. The decline in ΔH owing to the incomplete gelatinization in the condition where water mobility is restricted[53].

      Moreover, xanthan with sodium alginate through the interaction with the maize starch granules formed a hydration film via hydrogen bonding cross-linking and/or coating retard normal corn starch gelatinization[54].

      Therefore, we summarized the results of NSPs from different origins on starch gelatinization properties in Table 2. Most studies give the results that hydrocolloids lead Tc increased or unchanged while To remain unchanged or increased. Among most research, ΔH value was found to be decreased while the phase-transition temperature range was varied across the literature.

      Table 2.  Effect of non-starch polysaccharides on starch gelatinization.

      Type of non-starch polysaccharideType of starchToTpTcΔHReference
      Botanical
      ArabinoxylansWheat starch↑/↓ (depends on arabinoxylans molecular weight)↑/—↑/↓/——[28]
      β-glucansRice starch↑↑↑↓[25]
      Corn fiber gumWheat starch———↑[29]
      Carboxymethyl celluloseWheat starch↑N↑↑[23]
      Carboxymethyl celluloseNixtamalization maize dough↑↑↑↓[24]
      Fenugreek gumCorn starch↑/↓(depends
      on starch
      nitrationation)      		NN—[31]
      Guar gumChestnut starch↑↓/—(depends
      on guar concentration)      		↑↑[38]
      Guar gumAcorn starch↑↑↑↓[40]
      InulinWheat starch↑↑↑/—(depends
      on inulin DP)      		↓/—(depends
      on inulin DP)      		[27]
      Konjac glucomannanCorn starch↑↑↑—[34]
      Konjac glucomannanPotato starch—↑↑↓[35]
      Konjac glucomannanMaize starch/potato starch———/↑(depends
      on starch origin)      		↓[36]
      Mesona chinensis polysaccharideWaxy maize starch/normal maize starch↑↑↑↓[44]
      Okra extractWheat starch/corn starch↑↑N↑(wheat starch)/ ↓(corn starch)[43]
      Pectin/InulinPotato starch↑(pectin)/↓(inulin)↑(pectin)/↑(inulin)↑(pectin)/ —(inulin)↓(pectin)/ —(inulin)[37]
      Sodium alginateWheat starch↑NN↓[48]
      TamarindWaxy/normal/high amylose corn starchN↑N↑[30]
      Yellow mustard mucilageWheat starch/rice starchN—N↑[42]
      Animal
      ChitosanMaize starch↑↑↑↓[45]
      Microbial
      XanthanRice starch———↓[50]
      Multiple types
      β-glucans (curdlan, oat, barley and yeast β-glucans)Rice starch———↓[26]
      Guar gum/xanthanTapioca starch↑(guar)—(guar)/
      ↑(xanthan)      		—↓[51]
      Guar gum /CMC/Xanthan gum/tapioca extracts/tamarind seeds extractsWaxy rice starch/non-waxy rice starch————[8]
      Konjac glucomannan/ CMC/chitosanCorn starch——(konjac glucomannan, CMC)/↑(chitosan)—↓(konjac glucomannan)/ ↑(CMC)/ —(chitosan)[46]
      Xanthan gum/Guar gumYam starch————[41]
      To, Tp, and Tc represent the gelatinization beginning, highest, and end temperatures, respectively. The ΔH represents enthalpy (the heat energy required by the test starch during the endothermic transition). The arrow (↑, ↓, —) represents an increase/decrease or a no change in temperature, respectively. The letter "N" represents the corresponding parameters not mentioned in the research.
    The mechanism of NSPs on starch gelatinization

      NSPs reduce the water activities for starch gelatinization

    • NSPs play a role in reducing water activity during starch gelatinization. When polysaccharides are added to starch, there is an observed increase in gelatinization temperature, especially with higher concentrations of polysaccharides. This delayed gelatinization occurs because the polysaccharides limit water availability[55], decreasing the number of water molecules accordingly. Water molecules' access to the starch interior is hindered, directly impacting the hydrogen bonds between them, thus restricting starch swelling ability[48].

      NSPs can interact with starch

    • NSPs, for example, glucomannan and xanthan will decrease the starch fluidity while β-glucans show a relatively weak influence[56]. Gelatinization endotherm refers to the energy required for starch granules to collapse and disassemble the molecular structure. The increase of ΔH owing to the starch chain limitation. Sodium alginate decreased ΔH of rice starch indicating a restricted gelatinization process and partial gelatinization of starch granules[57]. During heating, polysaccharides can act as a protective membrane, thus inhibiting starch expansion. However, at higher hydrocolloid concentrations, the hydrophilic chain between NSPs and starch might be conducive to the increase of ΔH. From the molecular scope to interpret this phenomenon, though the short-range order may not be changed by the NSPs, there are fewer double helix structures formed, which may be owing to the partly disruptive effect NSPs played on the original double helix structure in starch[58]. Consistent with the FTIR results, the NSPs can reduce the crystallinity of porous maize starch (XRD)[59]. The 13C NMR test also suggests that the single and double helix structure of the original starch changed differently according to the types of NSPs added[58]. Luo et al., also verified that NSPs modify the rearrangement of amylose especially the linear chains around the gelatinization molecules[48]. Therefore, the interactions between polysaccharide molecules and starch play a vital role in determining the gelatinization profile[56].

      NSPs show different effects on starch gelatinization temperatures and endotherm enthalpy, which delays the progress and incomplete gelatinization in most conditions, which will exert various significant influences on starch susceptibility to enzymatic digestion. As the gelatinization degree increased, the starch hydrolysis degree increased and vice versa[4]. Therefore, the next part will focus on the NSPs' role in the digestion of starch.

    Effect of NSPs on starch digestion

    • As mentioned above, NSPs can interact with starch so undoubtedly, they can play a critical role in determining starch digestibility. We have summarized the articles associated with non-starch polysaccharides on starch hydrolysis in recent years in Tables 3 & 4. Table 3 presented macroscopic profiles of starch hydrolysis caused by NSPs while Table 4 mainly focused on the changing trends in specific starch digestion parameters (such as rapidly digestible starch content, slowly digestible starch content, resistant starch content, starch equilibrium hydrolysis concentration, and hydrolysis reaction rate). From the Tables, it is clear to see, that most hydrocolloids cause a significant inhibition on kinds of starches, though their structure and functional abilities vary. Most non-starch polysaccharides reduced the RDS, except chitosan. Reports of the opposite trend were also given that for corn starch, NSPs, such as xanthan and guar gum, raised the content of RDS. In terms of RS, NSPs increased their amount, except for xanthan gum and chitosan. The impact of NSPs on starch digestion parameters (C∞ and k) was generally reduced, thereby bringing about the digestion inhibition effect on starch and the lowering effect on the glycemic index. From the above mentioned, NSPs undoubtedly strongly interfere with starch digestion, leading to a lower glycemic index. We summarize the main mechanism of action as follows (Fig. 2).

      Table 3.  Effect of non-starch polysaccharides on starch digestibility.

      Type of non-starch polysaccharideType of starchSome findings and conclusionsReference
      Psyllium(Gluten-free bread) RicePSY reduces the chickpea flour-based bread glycemic response.[86]
      Gellan gumRiceGellan gum reduced starch digestion and GI index.[87]
      Guar gum/sodium alginate xanthan gum/Waxy riceThe NSPs decreased the starch digestion rate.[88]
      Xanthan gumRiceXanthan increased the glycemic index of the mixture.[89]
      Nano-celluloseCornHigher nano-cellulose amounts slow down the initial glucose release rates.[90]
      Carboxymethyl cellulose/ xanthan gum/ guar gumFried-natural fermented
      rice noodles (rice)      		NSPs improve digestion.[72]
      psylliumRice /cassavaThe psyllium decreased starch digestion.[91]
      Nano-fibrillated celluloseCornNSPs reduced the level of hydrolysis glucose.[92]
      CMC/ guar/ xanthan gumHigh amylose riceNSPs decreased the surge of blood glucose.[93]
      PectinCornPectin hindered starch digestion.[62]
      ChitosanWaxy maizeChitosan modification altered starch digestion.[94]
      Guar/ xanthan gum/ sodium alginateWheat/buckwheatThe hydrocolloid's addition reduced starch hydrolysis.[95]
      Xanthan/ guar gum/ pectin/ konjac-glucomannanGelatinized potatoNSPs hindered starch digestion and the extent perform on blood glucose depends highly on the types.[96]
      Locust bean/ guar/ fenugreek/ xanthan/
      flaxseed gum      		CornThe XG showed a prominent effect in interfering with glucose.[97]
      Extracted malva nut gumWheat bread (wheat)MNG-containing breads showed low glucose levels.[98]

      Table 4.  Non-starch polysaccharides influence on RDS, SDS, RS and digestion parameters.

      Type of non-starch polysaccharideType of starchRDSSDSRSC∞ (equilibrium concentration)k (kinetic constant)Reference
      XanthanRice↓↑—↓↓[78]
      Creeping fig seed polysaccharidePotato↓↓↑↓↓[79]
      PectinCorn↓↑↑↓↓[80]
      Arabic/ xanthan/ guar gumCorn↓↓/↑(xanthan)↑/↓(xanthan)NN[7]
      Guar gumRice↓↑↑↓↓[8]
      Chitosan/ xanthan/
      sodium alginate      		Wet sweet potato↑(chitosan)/
      ↓(xanthan, SA)      		↑(chitosan, xanthan)/
      ↓(SA)      		↓(chitosan)/
      ↑(xanthan, SA)      		NN[81]
      Guar gumLotus seed↓—↑NN[68]
      Pullulan/pectinFried potato↓↑↑↓↓[60]
      Konjac glucomannanQuinoa/maize↓↓↑NN[82]
      ChitosanLotus seed↓↑—NN[68]
      Hydroxypropylmethyl cellulose (HPMC)/ carboxymethyl cellulose/ xanthan gum (XG)/ apple pectin (AP)gluten-free potato steamed bread(potato starch)↓↓↑↓↓[83]
      PectinCorn↓↑↑NN[84]
      Cellulose nanocrystalsCorn /pea /potato↓↓↑NN[85]
      PullulanRice↓↑↑↓↓[10]
      High methoxylated pectin/ guar gum/ carboxymethyl cellulose/ xanthan gum/ hydroxypropylmethyl celluloseCorn /potato↑/↓(guar gum in terms of potatoes starch)↓(corn starch)/ —(potatoes starch)—/↓(xanthan and HPMC in terms of corn starch)/
      ↑(potatoes starch exception of HPMC)      		↑(corn starch by adding CMC, potatoes starch by adding guar gum and pectin)/ ↓(xanthan in corn starch)N[11]
      The arrow (↑, ↓, —) represents an increase/decrease or a no change in temperature, respectively. The letter "N" represents the corresponding parameters not mentioned in the research.

      Figure 2. 

      Mechanisms from lowering effects of NSPs on starch digestion.

      Restricting starch granule breakdown and delaying starch gelatinization, retaining more intact structures for the protection of enzymatic digestion[60]

    • The starch digestion rate is influenced by starch gelatinization which has been widely reported. NSPs such as galactomannan restrict starch expansion leaving granule ghosts in the paste. The unable to fully gelatinization of granules in the presence of hydrocolloids is also linked to the limited water availability. This gives rise to resistance toward enzymes[61]. Tester & Sommerville illustrated that the inhibition profile was always greater at the gelatinization temperature for each kind of starch and at higher starch-to-water ratios, where higher temperatures promote extensive gelatinization and mask the decreasing effect of NSPs on starch hydrolysis[55].

      The bulk viscosity of hydrocolloids reduces starch accessibility to the enzyme[62]

    • NSPs raise the bulk viscosity of the substrate, limiting the enzyme's accessibility. For example, guar gum as a kind of thickening agent can decrease glucose levels[63]. One of the most important reasons is that NSPs increase the viscosity of the food matrix which can result in slowing gastric emptying, restricting the diffusion of substrate[61,64]. However, mixing at high speeds can negate the hindering effect[64]. Kim & White reported oat starch hydrolysis decreased as the β-glucan molecular weight increased[65]. Apart from the viscosity factor, the NSPs may perform another physical effect during starch digestion progress. The structural modifications to the food matrix may also be a response to the change in starch digestibility[66]. The NSPs can coat the granules by forming a physical barrier as evidenced by the CLSM technique which protects the starch from hydrolysis[67]. Different levels of additional inulin also caused a different matrix structure leading to modified starch hydrolysis. A denser gluten network appears for the 5% inulin of degree of polymerization 12−14 enriched sample, while the starch digestion increased with a higher level of inulin, causing an easily disrupted protein architecture[67].

      Interact with starch molecules to assemble more ordered structures[68]

    • The effect of NSPs interacting with the leaching of amylose is indicated by Ramirez et al.[69] by the change of the complex index. This means the molecular interactions occurred when NSPs occurred. NSPs changed the crystalline structure of starch. The increasing inulin strengthens the XRD peak. The higher crystallinity may be due to the preferable digestion of amorphous regions or the formation of more ordered areas during hydrolysis. The more perfect crystalline with the addition of inulin may also lower the digestibility of starch[67].

      Hydrocolloids interact with enzymes thus changing the enzyme conformation and/or hindering its accessibility[62]

    • NSPs exemplified as cellulose or nanocrystalline cellulose can interact with α-amylase, their binding role on the enzyme, leading to an inhibition of the enzyme activity which relies highly on the hydrocolloid surface, packing density, and its entrapment on the enzyme[70,71].

      Apart from above mentioned, different phenomena also occur when hydrocolloids exist. High methoxylated pectin, carboxymethyl cellulose, and xanthan gum lead to an increased trend of RDS as opposed to guar gum, while CMC can decrease the RS of corn starch. A similar result was observed when guar gum, as well as pectin added to potato starch[11]. The researchers suggest that the hydrocolloid's origin plays a critical role in determining starch digestion and some polysaccharides may retard starch retrogradation, especially amylose as a result of the NSPs-amylose interaction. The basis lies in the composite network between the participants, and phase separation may occur. Besides, xanthan added to rice noodles brings air cells thus leading to higher water absorption and can promote the digestive enzymes' contact with starch inside areas and increase the rate of starch[72].

    Conclusions

    • The role of NSPs in the gelatinization properties of starch varies across the characteristics of NSPs and the types of starches. We classified NSPs into three major categories. In plant and animal sources, most NSPs increase the To and Tp of starch gelatinization. However, polysaccharides derived from microorganisms, such as xanthan gum, did not show an evident effect, while the mixture was more sensitive to salt. Most non-starch polysaccharides reduced the RDS, except chitosan. Reports of the opposite trend go for corn starch, NSPs, for example, xanthan and guar gum, raised the content of RDS. NSPs origin from botanica increased RS amount, except for xanthan gum and chitosan which are animal resources. First, some NSPs reduce water activity, due to their excellent hydration properties, thereby limiting starch gelatinization. Secondly, gums can interact with starch molecules (amylose or amylopectin), affecting the thermodynamic properties of the latter. As a consequence, the digestion of incompletely gelatinized starch-based foods is altered. From the perspective of reaction kinetics, the hydrolysis rate and final digestion starch concentration are altered, potentially influencing the physiological role of starch-based food by reducing glucose released into the bloodstream and affecting insulin levels. NSPs, with different origins, will exert distinct effects due to various properties (polysaccharide concentration, molecular weight, water holding capacity, charge, etc.) and used levels. In addition to the above-mentioned factors, the formation of interpenetrating network structure or the phase separation between NSPs with starch emerges, and the combination between NSPs and enzyme molecules affects the hydrolysis of starch as well.

      The current review only macroscopically summarizes the impact of various NSPs on starch gelatinization and digestibility, without intricately refining the structural characteristics of each colloid such as molecular weight, branching degree, molecular flexibility, charge positive or negative, and charge amount effect on starch. The critical or fundamental mechanisms by which NSPs affect starch properties are not identified, while aspects of mechanisms are generally covered. Furthermore, food, as a complex system, does not merely contain NSPs. The appearance of other components will also interfere with the starch, such as salt, protein, lipids, phenolic compounds, etc. To achieve a more comprehensive understanding of starch-based foods, the comprehensive effects of these aspects need to be further evaluated. Further research is necessary to deepen our understanding of these complex interactions and their implications for utilization.

    Author contributions

    • The authors confirm contribution to the paper as follows: writing-original draft: Li S; writing-review & editing: Li S, Zongo AWS, Chen Y, Liang H; resources: Li B; visualization: Li S, Chen W; validation: Li S, Chen Y; data curation: Li S, Chen W; form analysis: Li S, Li J, Liang H; project administration: Li J, Li B; supervision & funding acquisition: Li B. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All data generated or analyzed during this study are included in this published article.

      Acknowledgments

      • This work was supported by the National Key R&D Program of China (2022YFD2101300) & National Natural Science Foundation of China (Grant No. 32172200).

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Rights and permissions
      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press on behalf of China Agricultural University, Zhejiang University and Shenyang Agricultural University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (2)  Table (4) References (98)
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    Cite this article
    Li S, Chen W, Zongo AWS, Chen Y, Liang H, et al. 2023. Effects of non-starch polysaccharide on starch gelatinization and digestibility: a review. Food Innovation and Advances 2(4):302−312 doi: 10.48130/FIA-2023-0029
    Li S, Chen W, Zongo AWS, Chen Y, Liang H, et al. 2023. Effects of non-starch polysaccharide on starch gelatinization and digestibility: a review. Food Innovation and Advances 2(4):302−312 doi: 10.48130/FIA-2023-0029
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  • Introduction
  • Effect of NSPs on starch gelatinization

  • Starch gelatinization

  • NSPs

  • NSPs of botanic origin

  • Cellulose derivatives

  • Non-cellulose categories

  • β-glucans
  • Inulin
  • Arabinoxylans
  • Tamarind
  • Fenugreek gum
  • Konjac glucomannan
  • Guar gum
  • Carrageenan and Alginate

  • Pectin substances categories

  • Pectin polysaccharides
  • Extracts or mucilage
  • NSPs of animal origin

  • Chitosan

  • NSPs of microbial origin

  • Xanthan

  • The mechanism of NSPs on starch gelatinization

  • NSPs reduce the water activities for starch gelatinization

  • NSPs can interact with starch

  • Effect of NSPs on starch digestion

  • Restricting starch granule breakdown and delaying starch gelatinization, retaining more intact structures for the protection of enzymatic digestion[60]

  • The bulk viscosity of hydrocolloids reduces starch accessibility to the enzyme[62]

  • Interact with starch molecules to assemble more ordered structures[68]

  • Hydrocolloids interact with enzymes thus changing the enzyme conformation and/or hindering its accessibility[62]

  • Conclusions
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
  • About this article
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