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MAP  >  Food Materials Research
2023 Volume 3
Article Contents
Abstract
Introduction
Materials and methods
Plant materials
Protein extraction
Trypsin digestion
Affinity enrichment of ubiquitinated peptides
LC-MS/MS and data analysis
Bioinformatics methods
Results
Overview of the ubiquitomic analysis
Properties of ubiquitinated proteins and motifs
A temperature-regulated rice endosperm ubiquitome
Functions for differentially expressed ubiquitinated proteins
Discussion
Ubiquitination of heat shock proteins and transcription factors under high-temperature stress
Sucrose and starch metabolism are modified by ubiquitination under high-temperature stress
Ubiquitination affects sucrose hydrolysis
Ubiquitination of ADP-glucose pyrophosphorylase (AGPase)
Ubiquitination regulates amylose biosynthesis
Ubiquitination regulates amylopectin biosynthesis
Ubiquitination affects ribosomal systems under high-temperature stress
Author contributions
Data availability
Acknowledgments
Conflict of interest
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References
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ARTICLE   Open Access    

Effect of hydrogel/oleogel ratio, speed and time of mixing, on the mechanical properties of bigel materials and the application of Cox-Merz rule

  • 1.

    TecNM/Instituto Tecnológico de Durango, Blvd Felipe Pescador 1830 Ote, Col Nueva Vizcaya, Durango, Dgo., CP 34080, México

  • 2.

    Facultad de Ciencias Químicas, UASLP, Dr Manuel Nava, Zona Universitaria, San Luis Potosí, S, L.P., México

More Information
  • Bigels, are materials that combine hydrogels and oleogels. However, a major problem with them is the characterization of a single material compound by two materials that have different physical characteristics. The objective of this research was to evaluate the effect of the mixing time, the interaction of ratio hydrogel/oleogel, mixing rate and the applicability of Cox-Merz rule in bigels. Bigels were obtained blending monoglycerides, canola oil for oleogel phas, esters of polyglycerol with linearly esterified polyricinoleic acid, Polyoxyethylene sorbitan monostearate (Tween 60), guar gum and water. The bigels were prepared by incorporating the oleogel into the hydrogel and mixed at different ratios (67:33, 83:17; 85:15), followed by stirring at two different rate (600 and 800 rpm) for 20 min. Firmness was evaluated for all experimental samples. Thermal scanning was done by DSC. Steady Shear and Oscillatory test were carried out on the samples. Creep- Compliance test, and Cox-Merz rule were used for data analysis. Texture analysis indicated that a higher level of oleogel in the bigel, diminished firmness, however, major differences were observed between samples at low rate of agitation and high time of mixing. The creep test was modeled by Burgers model with good fit (R2 > 0.9). High recovery (66–67%) was observed at lower presence of oleogels and high mixing rate. Data of steady shear and oscillatory shear were adjusted to empirical Cox-Merz rule with good fit at low shear rate (> 10 s−1).

  • Introduction

    Rice (Oryza sativa L.) is a world staple crop that feeds over half of the world's population[1,2]. In recent years, high-temperature events are becoming more frequent and intensive as a result of global warming, which can severely affect rice grain yield and quality[3,4]. During flowering and grain filling stages, high-temperature stress can result in a significant reduction in seed setting rate and influence amylose content, starch fine structure, functional properties and chalkiness degree of rice[5−7]. Transcriptome and proteome analysis in rice endosperm have also been used to demonstrate the differences in high-temperature environments at gene and protein expression levels[8−10]. In addition, as an important post-translational modification, protein phosphorylation has proven to be involved in the regulation of starch metabolism in response to high-temperature stress[11]. However, little is known about whether protein ubiquitination regulates seed development under high-temperature stress.

    Ubiquitination is another form of post-translational modification that plays key roles in diverse cellular processes[12]. Several reports have described the functions of ubiquitination in rice defense responses based on ubiquitome analysis. Liu et al.[13] investigated relationships between ubiquitination and salt-stress responses in rice seedlings using a gel-based shotgun proteomic analysis and revealed the potential important role of protein ubiquitination in salt tolerance in rice. Xie et al.[14] identified 861 peptides with ubiquitinated lysines in 464 proteins in rice leaf cells by combining highly sensitive immune affinity purification and high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). These ubiquitinated proteins regulated a wide range of processes, including response to stress stimuli. A later study revealed the relationships between ubiquitination status and activation of rice defense responses, and generated an in-depth quantitative proteomic catalog of ubiquitination in rice seedlings after chitin and flg22 treatments, providing useful information for understanding how the ubiquitination system regulates the defense responses upon pathogen attack[15]. Although many studies have shown that ubiquitination plays improtant roles in the heat response of plant[16,17], there has been little systematic discussion on the ubiquitome of rice endosperm in the context of global climate change.

    In this study, we examine the high-temperature induced ubiquitination change in two rice varieties with different starch qualities, through a label-free quantitative ubiquitome analysis. This study provides a comprehensive view of the function of ubiquitination in high-temperature response of rice developing seed, which will shed new light on the improvement of rice grain quality under heat stress.

    Materials and methods

    Plant materials

    Two indica rice varieties with different starch quality, 9311 and Guangluai4 (GLA4), were used as materials. 9311 is a heat-sensitive variety, which displays low amylose content with good starch quality; while GLA4 is known to be the parental variety of HT54, an indica breeding line with heat tolerance, and thus GLA4 is possibly heat tolerant, which shows high amylose content with poor starch quality[18,19]. Rice growth conditions, sample treatment and collection were conducted as previously described[11].

    Protein extraction

    Husk, pericarp and embryo were detached from immature rice grains on ice[20]. Rice endosperm was then ground with liquid nitrogen, and the cell powder was sonicated 10 rounds of 10 s sonication and 15 s off-sonication on ice in lysis buffer (6 M Guanidine hydrochloride, pH 7.8−8.0, 0.1% Protease Inhibitor Cocktail) using a high intensity ultrasonic processor. Following lysis, the suspension was centrifuged at 14,000 g for 40 min at 4 °C to remove the debris. The supernatant was collected, and the protein concentration was estimated using BCA assay (Pierce BCA Protein assay kit, Thermo Fisher Scientific, Waltham, MA, USA) before further analysis.

    Trypsin digestion

    The protein mixture was reduced by DTT with the final concentration of 10 mM at 37 °C for 1 h, alkylated by iodoacetamide with a final concentration of 50 mM at room temperature in the dark for 0.5 h, and digested by trypsin (1:50) at 37 °C for 16 h. Then the sample was diluted by adding trifluoroacetic acid (TFA) to the final concentration of 0.1%. The enzymatic peptides were desalted on a Sep-Pak C18 cartridge (Waters, Milford, MA, USA), concentrated by lyophilization and reconstituted in precooled IAP buffer (50 mM MOPS-NaOH PH 7.2, 10 mM Na2HPO4, 50 mM NaCl) for further analysis.

    Affinity enrichment of ubiquitinated peptides

    The peptides solution was incubated with prewashed K-ε-GG antibody beads (PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit), and gently shaken at 4 °C for 1.5 h. The suspension was centrifuged at 2,000 g for 30 s, and the supernatant was removed. The Anti-K-ε-GG antibody beads were washed with IAP Buffer three times and with ddH2O three times. The peptides were eluted from the beads with 0.15% trifluoroacetic acid (TFA). Finally, the eluted fractions were combined and desalted with C18 Stage Tips.

    LC-MS/MS and data analysis

    LC-MS/MS analysis were performed using the methods of Pang et al.[11]. Raw mass spectrometric data were analyzed with MaxQuant software (version 1.3.0.5) and were compared with the indica rice protein sequence database (Oryza sativa subsp. indica-ASM465v1). Parameters were set according to Pang et al.[11]. All measurements were obtained from three separate biological replicates.

    Bioinformatics methods

    Quantification of the modified peptides was performed using the label-free quantification (LFQ) algorithm[11]. Differentially ubiquitinated sites (proteins) in response to high-temperature were identified by Student's t-test (p < 0.05, log2(fold-change) > 1) with at least two valid values in any condition or the ubiquitination sites that exhibited valid values in one condition (at least two of three replicates) and none in the other.

    Subcellular localization was performed using CELLO database (http://cello.life.nctu.edu.tw). Gene Ontology (GO) annotation proteome was derived from the AgriGO (http://bioinfo.cau.edu.cn/agriGO/). The differential metabolic profiles were visualized with MapMan software (version 3.6.0RC1). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation was performed by using KEGG Automatic Annotation Server (KAAS) software. A p-value of < 0.05 was used as the threshold of significant enrichment. SWISS-MODEL was used to generate the tertiary structure of GBSSI (SWISS-MODEL, http://swissmodel.expasy.org/). The figures were annotated with Adobe Illustrator (Adobe Systems, San Jose, CA, USA).

    Results

    Overview of the ubiquitomic analysis

    To elucidate how high-temperature stress influences rice developing endosperm at the ubiquitination level, a label-free analysis was performed to quantify ubiquitome from two indica rice varieties under normal (9311-C and GLA4-C) and high-temperature conditions (9311-H and GLA4-H). The distribution of mass error of all the identified peptides was near zero and most of them (71.5%) were between -1 and 1 ppm, suggesting that the mass accuracy of the MS data fits the requirement (Fig. 1a). Meanwhile, the length of most peptides distributed between 8 and 42, ensuring that sample preparation reached standard conditions (Fig. 1b).

    Figure 1.  Characteristics of the ubiquitinated proteome of rice endosperm and QC validation of MS data. (a) Mass error distribution of all identified ubiquitinated peptides. (b) Peptide length distribution. (c) Frequency distribution of ubiquitinated proteins according to the number of ubiquitination sites identified.
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    In all endosperm samples, a total of 437 ubiquitinated peptides were identified from 246 ubiquitinated proteins, covering 488 quantifiable ubiquitinated sites (Supplemental Table S1). Among the ubiquitinated proteins, 60.6% had only one ubiquitinated lysine site, and 18.7%, 8.1%, 5.3%, or 7.3% had two, three, four, or five and more ubiquitinated sites, respectively. In addition, four proteins (1.6%, BGIOSGA004052, BGIOSGA006533, BGIOSGA006780, BGIOSGA022241) were ubiquitinated at 10 or more lysine sites (Fig. 1c, Supplemental Table S1). The proteins BGIOSGA008317 had the most ubiquitination sites with the number of 16. It was noted that besides ubiquitin, two related ubiquitin-like proteins NEDD8 and ISG15 also contain C-terminal di-Gly motifs generated by trypsin cleavage, and the modifications of these three proteins cannot be distinguished by MS[21]. Here, the di-Gly-modified proteome therefore represents a composite of proteins modified by these three proteins. However, the sites from NEDD8 or ISG15 modifications were limited because they mediate only a few reactions in cells[21].

    Properties of ubiquitinated proteins and motifs

    To better understand the lysine ubiquitome changes in rice endosperm induced by high-temperature, we performed a Gene Ontology (GO) functional annotation analysis on all identified ubiquitinated proteins (Fig. 2a). In the biological process GO category, 'metabolic process' and 'cellular process' were mainly enriched, accounting for 75.1% and 74.1% of ubiquitinated proteins, respectively. In addition, 34.6% proteins were associated with 'response to stimulu', emphasizing the regulatory role of ubiquitination modification in response to high-temperature stress. From the cellular component perspective, ubiquitinated proteins were mainly associated with 'cellular anatomical entity' (99.4%), 'intracellular' (84.4%) and 'protein-containing complex' (29.9%). The molecular function category result suggested that these proteins were largely involved in 'binding' (62.7%), 'catalytic activity' (43.4%) and 'structural molecule activity' (16.5%). Furthermore, subcellular location annotation information indicated that 34.7%−39.4% proteins were located in the cytoplasm, and other were mostly located in the nucleus (23.5%−27.7%), plasma membrane (9.4%−11.4%), and chloroplast (9.6%−12.8%) (Fig. 2b). It is noteworthy that the ubiquitinated proteins located in the cytoplasm were decreased in high-temperature environments in both varieties.

    Figure 2.  Analysis of ubiquitinated proteins and motifs. (a) Gene ontology (GO) functional characterization of ubiquitinated proteins. (b) Subcellular localization of ubiquitinated proteins. From the inside out, the ring represents 9311-C, 9311-H, GLA4-C and GLA4-H, respectively. (c) Motif enrichment analysis of ubiquitinated proteins.
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    The following two significantly enriched motifs from all of the identified ubiquitinated sites were identified using MoDL analysis: [A/S]xKub and Kubxx[E/Q/R/V]x[E/G/L/P/Q/R/Y], which covered 84 and 100 sequences, respectively (Fig. 2c). Further analysis showed that the conserved alanine (A) and glutamic acid (E) were included in upstream and downstream of the ubiquitinated lysine sites in rice endosperm. A similar phenomenon also occurred in rice leaf[14], wheat leaf[22], and petunia[23], indicating that alanine (A) and glutamic acid (E) were likely to be the specific amino acids in conserved ubiquitination motifs in plants. Additionally, serine (S) was enriched at the position -2 (upstream) of the ubiquitinated lysine, while various amino acids such as arginine (R), glutamic acid (E), glutamine (Q), valine (V) were found at positions +3 and +5 (downstream).

    A temperature-regulated rice endosperm ubiquitome

    To detect possible changes in rice endosperm ubiquitome attributable to high-temperature stress, we then performed LFQ analysis on all quantifiable ubiquitination sites within our dataset. As shown in Fig. 3a, more ubiquitinated proteins, peptides and sites were detected in the treatment groups (9311-H and GLA4-H), suggesting that exposure to high-temperature stress may increase the ubiquitination events in rice endosperm. Only 282 common ubiquitinated sites in 158 proteins were quantifiable for all sample groups due to reversible ubiquitination induced by high-temperature (Fig. 3b). Principal component analysis (PCA) showed that three repeats of each sample clustered together, and four groups were clearly separated (Fig. 3c). Furthermore, the differentially expression profiles of ubiquitination sites (proteins) in 9311 and GLA4 under high-temperature stress were depicted to further understand the possible changes (Fig. 3d). Where LFQ values were missing, the data were filtered to identify those ubiquitination sites with a consistent presence/absence expression pattern. These analyses yielded 89 ubiquitination sites that were only present in 9311-H and six that were only present in 9311-C (Fig. 3d, Supplemental Table S2). Similarly, 51 differentially expressed ubiquitination sites were present in GLA4-H and 13 ubiquitination sites only occurred in GLA4-C (Fig. 3d & Supplemental Table S3). Beyond that, a total of 113 and 50 significantly changed ubiquitination sites (p < 0.05, log2(fold-change) >1) were screened out in 9311 and GLA4, respectively (Fig. 3d, Supplemental Tables S4 & S5). For subsequent comparative analysis, the ubiquitination expression profiles with consistent presence/absence and ubiquitination sites with significant differences in statistical testing were combined and named as 9311-Up, 9311-Down, GLA4-Up, and GLA4-Down, respectively (Fig. 3d). The number of significantly up-regulated ubiquitination sites was far greater than down-regulated ubiquitination sites in both 9311 and GLA4 varieties. These findings indicated that high temperature not only induced the occurrence of ubiquitination sites, but also significantly upregulated the intensity of ubiquitination. Beyond that, the magnitude of the up-regulation in 9311 was higher than that in GLA4 (Fig. 3b & d), indicating that the ubiquitination modification of heat-sensitive varieties was more active than heat-resistant varieties in response to high-temperature stress.

    Figure 3.  A temperature regulated rice endosperm ubiquitome. (a) The number of ubiquitinated proteins, peptides and sites detected in four group samples. (b) Venn diagram of ubiquitination sites (proteins) detected in four group samples. (c) PCA based on ubiquitination intensity across all four sample groups with three biological repetitions. (d) Differentially expression profiles of ubiquitination sites (proteins) in 9311 and GLA4 under high-temperature stress. The expression profiles of selected ubiquitination sites (p < 0.05, log2(fold-change) >1) were normalized using the Z-score and presented in a heatmap.
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    To further investigate the ubiquitination regulatory pattern under high temperature stress in two varieties, four groups with significantly regulated sites were analyzed. There were 37 ubiquitination sites showed the same regulatory trend in 9311 and GLA4, accounting for 17.8% and 32.5% of the total differentially expressed sites in 9311 and GLA4, respectively. Among them, 36 ubiquitination sites were upregulated and one site was downregulated (Fig. 4a). In addition, 159 upregulated ubiquitination sites and three downregulated sites were only present in 9311, while 53 upregulated sites and 15 downregulated sites were only present in GLA4. Moreover, nine ubiquitination sites showed opposite regulatory trends in 9311 and GLA4. A similar regulatory trend of ubiquitination proteins is shown in Fig. 4b. It is noted that some proteins had both upregulated and downregulated ubiquitination sites (Supplemental Tables S6 & S7), indicating that significant differences in ubiquitination were, to some extent, independent of protein abundance.

    Figure 4.  Comparison of differentially ubiquitinated sites and proteins in 9311 and GLA4 under high-temperature stress.
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    Functions for differentially expressed ubiquitinated proteins

    To understand the function of ubiquitination in response to the high-temperature stress of rice endosperm, we conducted GO enrichment-based clustering analysis of the differentially ubiquitinated proteins in 9311 and GLA4 at high temperature, respectively (Fig. 5). In the biological process category of 9311, proteins were relatively enriched in the carbohydrate metabolic process, polysaccharide metabolic process, starch biosynthetic process, cellular macromolecule localization, protein localization, intracellular transport, and phosphorylation (Fig. 5). For the molecular function analysis, we found that the proteins related to kinase activity, nucleotidyltransferase activity, phosphotransferase activity, and nutrient reservoir activity were enriched (Fig. 5). The two principal cellular components were intrinsic component of membrane and integral component of membrane (Fig. 5). There was no significantly enriched GO term in the GLA4 group due to the dataset containing relatively few proteins, and thus, further enrichment analysis was conducted on the proteins that were common to both varieties. The results showed that proteins were over-represented in carbon metabolism, including starch biosynthesis and metabolism, glucan biosynthesis and metabolism, and polysaccharide biosynthesis and metabolism (Fig. 5), indicating the importance of carbohydrate synthesis and metabolism in the ubiquitination regulatory network.

    Figure 5.  Enrichment analysis of differentially expressed ubiquitinated proteins based on Gene Ontology (GO) terms.
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    To identify pathways which were differentially ubiquitinated under high-temperature stress, the KEGG pathway-based clustering analysis was conducted. The results showed that the differentially ubiquitinated proteins in both 9311 and GLA4 were mostly abundant in the pathways of carbohydrate metabolism, starch and sucrose metabolism, folding, sorting and degradation, translation, ribosome, and protein processing in endoplasmic reticulum (Fig. 6a). In the 9311 group, the pathways of carbohydrate metabolism, starch and sucrose metabolism, glycosyltransferases, glycolysis, and energy metabolism were enriched in the differentially ubiquitinated proteins (Fig. 6b); while there was no significantly enriched KEGG pathway in the GLA4 group. We further found the proteins that were common to both varieties were only significantly enriched in the starch and sucrose metabolism pathways (p = 0.04). The ubiquitination proteins involved in the starch and sucrose metabolism mainly include: sucrose hydrolysis (SUS, FK, UGPase), and starch synthesis (AGPase, GBSSI, BEI, BEIIb, PUL, PHO1), which are discussed below.

    Figure 6.  KEGG classification and enrichment analysis of differentially ubiquitinated proteins. (a) Number of differentially ubiquitinated proteins based on KEGG classification in 9311 and GLA4. (b) KEGG enrichment analysis of differentially ubiquitinated proteins in 9311.
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    Discussion

    Although many reports have described specific examples of ubiquitination in rice defense responses[13,15,16], our knowledge on global changes in the developing endosperm ubiquitome under high-temperature stress is still lacking. In this study, a label-free quantitative proteomic analysis of ubiquitination was applied to examine the high-temperature induced ubiquitination change of two indica rice varieties (9311 and GLA4) with distinct starch quality. We identified many new lysine modification sites on proteins involved in various pathways, highlighting the complexity of the ubiquitination-mediated regulatory system in high-temperature stress responses in rice.

    Ubiquitination of heat shock proteins and transcription factors under high-temperature stress

    Heat shock proteins accumulate under various stresses and play important roles in plant defenses against abiotic stresses[24,25]. Research has shown that a number of heat shock proteins were prominent in the rice ubiquitome network, of which OsHSP71.1, and OsHSP82A showed increased ubiquitination levels under chitin and flg22 treatment[15]. Here, seven lysine residues on five heat shock proteins possessed ubiquitination modification in rice endosperm. Three sites (BGIOSGA011420-K78, BGIOSGA026764-K99, BGIOSGA029594-K106) showed significant up-regulation in 9311 under high-temperature stress, while in GLA4, the ubiquitination level of BGIOSGA011420-K78 was down-regulated. This differential ubiquitination of heat-tolerant and heat-sensitive varieties provided a basis for studying the regulation of post-translational modification of heat shock proteins under high-temperature stress, despite the regulatory role of those heat shock proteins being still unclear.

    Transcription factors (TFs) play an essential role in the regulation of gene expression. A total of three transcription factors were identified in the ubiquitination dataset, of which two were NAC family members. As one of the largest plant-specific TF families, NAC is involved in the responses to abiotic and biotic stresses[26]. The ubiquitination modification of K173 in BGIOSGA018048 was specifically expressed in the 9311-H group, which may affect the stress resistance level in high-temperature environments. In addition, two sites K148 and K149 of ERF TF family member BGIOSGA024035 was downregulated in GLA4 under high-temperature stress. This differential ubiquitination was likely to affect the expression of related genes regulated by this transcription factor.

    Sucrose and starch metabolism are modified by ubiquitination under high-temperature stress

    Ubiquitination affects sucrose hydrolysis

    The results of GO and KEGG enrichment analysis of differential ubiquitination proteins indicated that the sucrose and starch metabolic pathway was largely affected by ubiquitination regulation under high-temperature stress (Figs 5 & 6). The ubiquitination sites involved in sucrose and starch metabolism are listed in Table 1. To assess how high-temperature stress affects the crucial pathway, the significantly differential ubiquitination sites in 9311 and GLA4 were displayed in the heatmap of specific proteins (Fig. 7).

    Table 1.  Ubiquitination sites related to sucrose and starch metabolism in rice endosperm.
    Gene nameAnnotationProtein entryModification site(s)
    SUS1Sucrose synthase 1BGIOSGA010570K172, K177
    SUS2Sucrose synthase 2BGIOSGA021739K160, K165, K176, K804
    SUS3Sucrose synthase 3BGIOSGA026140K172, K177, K541, K544, K588
    FKFructokinaseBGIOSGA027875K143
    UGPaseUDP-glucose pyrophosphorylaseBGIOSGA031231K27, K150, K303, K306
    AGPS1ADP-glucose pyrophosphorylase small subunit 1BGIOSGA030039K94, K464, K484
    AGPS2ADP-glucose pyrophosphorylase small subunit 2BGIOSGA027135K106, K132, K385, K403, K406, K476, K496
    AGPL2ADP-glucose pyrophosphorylase large subunit 2BGIOSGA004052K41, K78, K134, K191, K227, K254, K316, K338, K394, K396, K463, K508, K513
    AGPL3ADP-glucose pyrophosphorylase large subunit 3BGIOSGA017490K509
    GBSSIGranule bound starch synthase IBGIOSGA022241K130, K173, K177, K181, K192, K258, K371, K381, K385, K399, K462, K517, K530, K549, K571, K575
    BEIStarch branching enzyme IBGIOSGA020506K103, K108, K122
    BEIIbStarch branching enzyme IIbBGIOSGA006344K134
    PULStarch debranching enzyme:PullulanaseBGIOSGA015875K230, K330, K431, K736, K884
    PHO1Plastidial phosphorylaseBGIOSGA009780K277, K445, K941
     | Show Table
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    Figure 7.  Sucrose and starch pathway at the ubiquitination levels in rice endosperm under high-temperature stress.
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    In cereal endosperm, sucrose is the substrate for the biosynthesis of starch. The formation of glucose 1-phosphate (G1P, used in starch synthesis, see below) from sucrose requires a series of enzymes[27]. Here, we found that sucrose synthase 1 (SUS1), SUS2, SUS3, fructokinase (FK), and UDP-glucose pyrophosphorylase (UGPase) were ubiquitinated among all sample groups (Table 1, Fig. 7). In the ubiquitome of seedling and leaf in japonica rice, ubiquitination sites have been found in SUS1, SUS2, UGPase, and FK, which were related to sucrose hydrolysis[14,15]. SUS catalyzed the process of cleaving sucrose into UDP-glucose (UDPG) and fructose. Two ubiquitination sites, K172 and K177, were identified in SUS1 in rice endosperm, which were also found in rice leaves[14]. A total of four ubiquitination sites were identified in SUS2, two of which were also reported in rice seedling and leaf, indicating the conservation of the lysine residues in different rice tissues. It was noted that all four ubiquitination sites in SUS2 were upregulated in high-temperature environments, although the regulated sites of 9311 and GLA4 were different. In 9311, the ubiquitination levels of K160, K174, and K804 were increased, while GLA4 was only upregulated in K176. The ubiquitination sites K541, K544, and K588 in SUS3 were screened from developing rice seeds for the first time. In addition, SUS3 had two completely overlapping sites K172 and K177 with SUS1, and it was difficult to determine which enzymes the two sites belonged to. The ubiquitination levels of SUS3-K541 and SUS3-K544 in 9311 significantly increased in high-temperature environments, while there was no significant difference in the ubiquitination level of SUS3 in GLA4. Overall, the ubiquitination sites of SUS in rice endosperm were located in the functional domain except for SUS2-K804, reflecting the importance of ubiquitination regulation in SUS.

    UGPase catalyses the conversion of glucose 1-phosphate and UTP into UDPG[28]. Research has shown that the mutation of UGPase gene lead to chalky endosperm[29]. As shown in Table 1, four ubiquitination sites K27, K150, K303, and K306 were identified in rice endosperm, which were completely inconsistent with the seven ubiquitination sites in rice seedlings and two in leaves[14,15], reflecting the tissue specificity. We speculated that the UGPase with different modification sites may play different regulatory roles in metabolic pathways in different tissues. Under high-temperature stress, the ubiquitination level of UGPase-K27 was 8.1-fold up-regulated. Liao et al.[10] demonstrated that the expression of UDPase was down-regulated in both heat-tolerant and heat-sensitive rice lines under high temperature conditions, which could reasonably explain the significant up-regulation of UGPase-K27 ubiquitination level. The ubiquitination site K143 of FK was also reported in seedling tissues[15].

    Ubiquitination of ADP-glucose pyrophosphorylase (AGPase)

    The AGPase reaction represents the first committed step of starch biosynthesis[27]. A total of 22 lysine ubiquitination sites were identified in four AGPase subunits (AGPL2, AGPL3, AGPS1, AGPS2). AGPL2 had 13 ubiquitination sites, of which six were located in NTP_transferase domain, including K254, K338, K191, K134, K227, and K316. High-temperature stress resulted in an increase in the ubiquitination level of K254 in both 9311 and GLA4, and significant upregulation of K508 and K513 in 9311, as well as K191, K227, and K316 in GLA4. In contrast, AGPL2-K394 were significantly downregulated in GLA4. AGPL3 contained one ubiquitination site K509, and the modification level of AGPL3-K509 was up-regulated in high-temperature environments in 9311. AGPS1 had one specific ubiquitination site K464 and another two sites K94 and K484 that completely overlapped with AGPS2-K106 and AGPS2-K496, respectively. The modification levels of K464 and K484 significantly increased in high-temperature environments in 9311, and K94 was significantly up-regulated in both varieties. There were seven ubiquitination sites in AGPS2 in rice endosperm, which were different with the sites found in rice leaves[14]. In addition to the two sites that overlapped with AGPS1, AGPS2 had another two ubiquitination sites (K406 and K132) that upregulated in high-temperature environments.

    Ubiquitination regulates amylose biosynthesis

    Amylose content is one of the key determinants that strongly influence rice grain quality[30]. The biosynthesis of amylose requires the catalytic effect of granule-bound starch synthase I (GBSSI)[30,31]. Here, a total of 16 ubiquitination sites were identified in GBSSI (Table 1, Fig. 8a). Among these ubiquitination sites, six lysine residues (K130, K173, K177, K181, K192, K258) were located in glycosyltransferase 5 (GT5) domain, and three sites (K399, K462, K517) were located in GT1 domain (Fig. 6), indicating the important role of ubiquitination regulation of GBSSI. Under high-temperature stress, the ubiquitination levels of six sites (K130, K177, K399, K381, K385, K549) increased in two indica rice varieties, while one sites (K258) showed downregulation in 9311 (Fig. 8a). Numerous studies had described that the amylose content was reduced under high-temperature stress in rice[5,7], which might be due to the degradation of GBSSI proteins caused by the increased significantly up-regulated ubiquitination sites. These ubiquitination sites identified in rice GBSSI with significant differences under high-temperature stress were expected to become a new breakthrough point for the improvement of starch quality.

    Figure 8.  Structure of GBSSI. (a) Domain structure of GBSSI and ubiquitination sites with significant differences in response to high-temperature stress. (b) 3D model of GBSSI and the relationship between ubiquitination sites K462 and ADP, SO4 (salt bridge or hydrogen bond).
    DownLoad: Full-Size Img PowerPoint

    To further determine the regulatory role of the ubiquitination sites in GBSSI, SWISS-MODEL was used to predict 3D structural model. As shown in Fig. 8b, GBSSI had three SO4 (sulfate ions) and one ADP ligand. These ligands interact with GBSSI through hydrogen bonds and salt bridges. Three sites, K447, R458, and K462, were associated with SO4 through salt bridges, while G100, N265, Q412, K462, and Q493 interact with the hydrogen bonds of ADP in GBSSI[32,33]. Based on this finding, it can be reasonably inferred that the K462 site with ubiquitination modification located in the GT1 domain played an important role in the interaction between GBSSI, SO4, and ADP. An in-depth investigation was necessary to gain a more comprehensive understanding of the regulatory function of ubiquitination modification at GBSSI-K462, although there was no significant difference in the ubiquitination level under high-temperature stress.

    Ubiquitination regulates amylopectin biosynthesis

    Amylopectin, the major component of starch, is synthesized by the coordinated action of multiple enzymes including soluble starch synthase (SSs), starch branching enzyme (BEs), starch debranching enzyme (DBEs), and phosphorylases (PHOs or Phos) with ADPG as a substrate. In this study, ubiquitination sites were detected in BEs, DBEs, and Phos.

    BEs, covering two isoforms, BEI and BEII, are responsible for catalyzing the formation of α-1,6-glucosidic linkages of amylopectin[34]. There were three ubiquitination sites (K103, K108, and K122) identified in BEI (Fig. 9a). K122 was the first amino acid in the carbohydrate-binding module 48 (CBM48) domain. Sequence alignment analyses of BEs from eight plants revealed K122 was conserved among all plants' BEI (Fig. 9a), suggesting a high probability of the functional effects of ubiquitination modification at this site. In high-temperature environments, ubiquitination levels of K108 and K122 were significantly up-regulated in 9311, while no significantly regulated ubiquitination sites of BEI were observed in GLA4. Only one ubiquitination site, K134, was found in BEIIb (Fig. 9a). The ubiquitination levels showed a slightly upward trend with no significant differences in high-temperature environments in both varieties. These changes could be one of the reasons for increased gelatinization temperature and relative crystallinity of rice starch in response to high-temperature[5].

    Figure 9.  Domain structure of (a) BEs, (b) PUL and (c) Pho1 as well as their ubiquitination sites with significant differences in response to heat stress. Residues in red indicate the ubiquitination site. Non-ubiquitinated residues are shown in dark grey.
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    DBEs consists of isoamylase (ISA) and pullulanase (PUL) with catalytic function for hydrolyzing α-1,6-glucosic linkages[35]. In the present study, we found that only PUL was ubiquitinated in rice endosperm (Fig. 9b). Among five ubiquitination sites (K230, K330, K432, K736, and K884) identified in PUL, K230 was located in the PULN2 domain, while K330 was in the CBM48 domain. Under high-temperature stress, K330 showed completely opposite regulatory trends in two cultivars. In addition, the ubiquitination level of K884, located in the DUF3372 domain, was significantly up-regulated in 9311. Previous study has reported that the expression of PUL was significantly up-regulated in 9311 under high-temperature stress, while GLA4 showed down-regulation in PUL abundance[11]. Consequently, there might be two possible functions of these ubiquitination sites. One possibility is that ubiquitination sites were unrelated to protein degradation; instead, they regulated the biosynthesis of amylopectin by affecting other functions of the protein. Secondly, ubiquitination sites were associated with protein degradation, and the levels of ubiquitination modification were based on protein abundance, resulting in a completely consistent regulation of ubiquitination modification and protein abundance under high-temperature stress.

    PHOs, including two types, Pho1/PHO1 and Pho2/PHO2, are responsible for the transfer of glucosyl units from Glc-1-P to the non-reducing end of a-1,4-linked glucan chains[36]. Pho1 is a temperature-dependent enzyme and considered crucial not only during the maturation of amylopectin but also in the initiation process of starch synthesis[37,38]. The three ubiquitination sites (K277, K445, K941) identified in Pho1 were located in two phosphorylase domains. We found that two sites, Pho1-K277 and Pho1-K445, were only ubiquitinated in high-temperature environments in 9311 and GLA4, respectively. Pang et al.[11] has demonstrated that the protein abundance of Pho1 decreased under high-temperature stress, especially in GLA4. Satoh et al.[38] reported that the functional activity of Pho1 was weakened under conditions of high temperature and its function might be complement by one or more other factors. Hence, these ubiquitination modifications that specifically occurred in high-temperature environments might be related to the degradation of Pho1 proteins.

    Ubiquitination affects ribosomal systems under high-temperature stress

    As a factory for protein synthesis in cells, the ribosome is an extremely crucial structure in the cell[39]. It has been proven that multiple ribosomal subunits were abundantly ubiquitinated in Arabidopsis and wheat[22]. In the present study, 57 ubiquitination sites involving 33 ribosome subunits were identified in 40S and 60S ribosome complexes in rice. Under high-temperature stress, the ubiquitination levels of some sites were significantly upregulated or downregulated, implying that ubiquitination of ribosomal proteins is likely to be an important regulatory mechanism in high-temperature response in rice endosperm. The results of GO and KEGG enrichment analysis indicated that the ribosome system was one of the most active systems for ubiquitination regulation under high-temperature stress. We speculated that the ubiquitin-proteasome system might be involved in the removal of subunits or entire ribosomes that were improperly folded in high-temperature environments. As shown in Fig. 10, the S10e, L18Ae, S27Ae, L9e, S3e, S28e, S20e, and S2e subunits were significantly up-regulated in 9311, while L13e subunits showed a completely opposite regulatory trend at the ubiquitination sites K81 and K88. In GLA4, the ubiquitination levels of S10e, S27Ae, L10Ae, L9e, S3e, S2e, and L4e showed a significant increase, while the ubiquitination level of L17e was significantly down-regulated under high-temperature stress. A total of seven ubiquitination sites involving S10e, S27Ae, L9e, S3e, and S2e subunits were jointly up-regulated in both two varieties. These sites might be related to the degradation of improperly folded ribosome subunits under high-temperature stress, while other ubiquitination sites with variety specificity might be associated with ribosomal function.

    Figure 10.  Ribosome system at the ubiquitination levels in rice endosperm under high-temperature stress. Grey shadings represent ubiquitinated proteins with no significant differences under heat stress. Red and blue shadings indicate up-regulated and down-regulated ubiquitinated proteins, respectively. Orange shading displays a combination of up- and down-regulated ubiquitinated sites in the same ubiquitinated protein.
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    In conclusion, this study provides the first comprehensive view of the ubiquitome in rice developing endosperm, and demonstrated that ubiquitination has diverse functions in the high-temperature response of rice endosperm by modulating various cellular processes, especially the sucrose and starch metabolism. Comparative analysis of the temperature-induced ubiquitination status revealed some similarities and more interesting differences between 9311 and GLA4. These differences might be the reason for the different qualities formation of the two indica rice varieties, which could provide potential genetic resources for the improvement of the heat resistance in rice. Considering the diversity of ubiquitination modification, it is worthwhile to further validate and explore the function and regulatory mechanism of the key targets and key pathways. The findings provide valuable insights into the role of ubiquitination in response to high-temperature stress and lay a foundation for further functional analysis of lysine ubiquitination in rice.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design: Bao J, Pang Y; data collection: Pang Y; analysis and interpretation of results: Pang Y; draft manuscript preparation: Ying Y; Revised manuscript preparation: Ying Y, Pang Y, Bao J. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    Acknowledgments

    This work was financially supported by the AgroST Project (NK2022050102) and Zhejiang Provincial Natural Science Foundation (LZ21C130003).

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

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  • Cite this article

    Mata-Mota JD, Gallegos-Infante JA, Pérez-Martínez JD, Rocha-Guzmán NE, González-Laredo RF. 2023. Effect of hydrogel/oleogel ratio, speed and time of mixing, on the mechanical properties of bigel materials and the application of Cox-Merz rule. Food Materials Research 3:24 doi: 10.48130/FMR-2023-0024
    Mata-Mota JD, Gallegos-Infante JA, Pérez-Martínez JD, Rocha-Guzmán NE, González-Laredo RF. 2023. Effect of hydrogel/oleogel ratio, speed and time of mixing, on the mechanical properties of bigel materials and the application of Cox-Merz rule. Food Materials Research 3:24 doi: 10.48130/FMR-2023-0024
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Effect of hydrogel/oleogel ratio, speed and time of mixing, on the mechanical properties of bigel materials and the application of Cox-Merz rule

  • 1.

    TecNM/Instituto Tecnológico de Durango, Blvd Felipe Pescador 1830 Ote, Col Nueva Vizcaya, Durango, Dgo., CP 34080, México

  • 2.

    Facultad de Ciencias Químicas, UASLP, Dr Manuel Nava, Zona Universitaria, San Luis Potosí, S, L.P., México

  • Received: 08 July 2023
  • Accepted: 28 August 2023
  • Published online: 11 October 2023
Food Materials Research  3,  Article number: 24  (2023)  |  Cite this article

Abstract: Bigels, are materials that combine hydrogels and oleogels. However, a major problem with them is the characterization of a single material compound by two materials that have different physical characteristics. The objective of this research was to evaluate the effect of the mixing time, the interaction of ratio hydrogel/oleogel, mixing rate and the applicability of Cox-Merz rule in bigels. Bigels were obtained blending monoglycerides, canola oil for oleogel phas, esters of polyglycerol with linearly esterified polyricinoleic acid, Polyoxyethylene sorbitan monostearate (Tween 60), guar gum and water. The bigels were prepared by incorporating the oleogel into the hydrogel and mixed at different ratios (67:33, 83:17; 85:15), followed by stirring at two different rate (600 and 800 rpm) for 20 min. Firmness was evaluated for all experimental samples. Thermal scanning was done by DSC. Steady Shear and Oscillatory test were carried out on the samples. Creep- Compliance test, and Cox-Merz rule were used for data analysis. Texture analysis indicated that a higher level of oleogel in the bigel, diminished firmness, however, major differences were observed between samples at low rate of agitation and high time of mixing. The creep test was modeled by Burgers model with good fit (R2 > 0.9). High recovery (66–67%) was observed at lower presence of oleogels and high mixing rate. Data of steady shear and oscillatory shear were adjusted to empirical Cox-Merz rule with good fit at low shear rate (> 10 s−1).

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    Introduction

    • Gels are defined as viscoelastic formulations; they can be made by hydro (water) or oleogels (organic liquid). Hydrogels have several advantages, such as their ease of preparation, non-oily nature, and for topical applications good spreadability, increased hydration, cooling effect, and ease of removal. The main drawback is that they only load hydrophilic compounds. On the other hand, oleogels are easy to obtain and can load hydrophobic compounds. The main problem of the use of oleogels in the food and pharmaceutical industry is their oily nature[1−3].

      The study of hydrogels and oleogels has been carried out for a long time, including several strategies to resolve problems, in this sense, the use of emulsion gel or emulgel has been introduced[4]. The emulgel usually is obtained by dispersing the liquid phase within the structured continuum phase[5]. The stability problem of the emulsion has been solved structuring disperse and continuum phase, this system has been named bigel[6]. Bigels have advantages of hydro and oleogels[7], also, the bigels, usually present better properties than either of the single gels[8] .

      Different combinations of hydrocolloids, gelators and vegetable oil has been studied, more relevant examples of used hydrocolloids are the use of Guar gum[9,10], gelatin[11,12], sodium alginate[13,14], starch[15,16] and synthetic polymers (Poly(vinyl alcohol) (PVA) and Polyvinylpyrrolidone (PVP)[17].

      Gelators for vegetable oil use sorbitan monopalmitate, policosanol, glyceryl stearate, beeswax, blend of monoglycerides[18,19], also, several vegetable oils have been used such as sunflower oil, olive oil and medium chain tryacylglycerides (MCT)[20, 21].

      Usually, rheological and microscopic techniques are used to describe the mechanical properties of the obtained bigels, also, the modelling of behavior has been carried out usually by evaluate cohesiveness, firmness, adhesiveness, stickiness, viscosity, and creep recovery of bigels[3,10], however, less attention has been paid to evaluate the process parameters as mixing power, mixing time; with only the of ratio hydrogel/oleogel being well described[2,14] evaluate the influence of ratio hydrogel/organogel and mixing rate, Zhu et al.[22] demonstrate that the increase of oleogel fraction in bigels increase the firmness. An interesting approach to study several materials is the Cox-Merz rule. The so-called Cox-Merz 'rule' is the empirical relationship which has been found to be of great use in rheology. It was observed by Cox & Merz[23] that for many polymeric systems, correspondence occurred between the steady state shear viscosity, η, plotted against shear rate, and the magnitude of the complex viscosity, |η*|, plotted against angular frequency, ω, however, this rule has not been applied in polymer systems with cross-linked behavior or gelled systems, but several authors have demonstrated that their applicability in gel-like emulsions has been suggested in the entanglement systems they obey the Cox-Merz rule, also, if the mechanical spectra shows a weak-gel behavior, the network can survive small deformations, before breaking structure[24,25], reported that several emulsions under specific conditions fit well with the Cox-Merz rule.

      To the best of our knowledge, no reports about the effect of mixing time and interaction of ratio hydrogel/organogel, mixing rate, mixing time and the applicability of Cox-Merz rule have been carried out for bigels.

    Materials and methods

    • Canola oil (61% monounsaturated, 32% polyunsaturated and 7% saturated) was purchased from the local supermarket (Durango, México) without further purification. Guar gum was purchased from Habacuq Comercializadora Química S.A de C.V. (Guadalajara, Jal, México). Myverol (18-04 PK) is a mixture of monoglycerides (mainly 49% glycerol monostearate, 48% glycerol monopalmitate and 3% calcium silicate) kindly provided by Kerry (SW food technology, SA de CV, Nuevo Leon, Mexico).

      The surfactant PGPR (E 476), which is a complex mixture of partial esters of polyglycerol with linearly esterified polyricinoleic acid derived from castor oil, was purchased from Palsgaard (San Luis Potosí, Mexico). Polyoxyethylene sorbitan monostearate (Tween 60) was purchased from Sigma-Aldrich (Toluca, México).

      Bigel preparation

    • Oleogels were prepared by dissolving myverol (10%, w/w) in canola oil (88.5% w/w) and PGPR as surfactant (1.5% w/w) under agitation at 80°C until complete solubilization following the method reported by Isaac Contreras-Ramírez et al[26]. The guar gum hydrogel (1%, w/w) was produced by dispersing the guar gum into distilled water under constant magnetic stirring at 80 °C for 1 h. Tween 60 was added to the water phase at 1.7% (w/w) under magnetic stirring. After that, the oil solution was cooled to 50 °C.

      The bigels were prepared by incorporating the oleogel into the hydrogel and mixed at different ratios (67:33, 83:17; 85:15), followed by stirring at two different rates (600 and 800 rpm) for 20 min with an Ultra Turrax homogenizer (T25, IKA, Germany). Then the mixtures were cooled to room temperature, to trigger the structuring of oil phase and water phase. The obtained bigels were subsequently stored at 4 °C for further analysis.

      Texture analysis

    • TA.TX plus texture analyzer (Texture Technologies Corp, New York, USA) was used to analyze the texture of the samples. Briefly, the instrument was calibrated (5 kg) and a penetration test was performed using a P20 cylindrical probe (aluminum probe, 20 mm diameter). A sample (50 g) of each formulation was placed in a container (35 mm diameter and 90 mm height) to a height of 35 mm. The probe penetrated at 5 mm·s−1 for 15 mm and returned to its initial position. From the force/time graph, the firmness value (maximum positive peak of the curve) was calculated following the method described by Pérez-Salas et al[27].

      Thermal analysis

    • The differential scanning calorimeter DSC (TA Instruments, DE, USA) was used to analyze the thermal properties of bigel samples. About 10–15 mg of each bigel sample was sealed in aluminum pans with lids, an empty aluminum pan was used as a control. The thermal profile was determined in the cycle heating/cooling temperature into the range of 20 to 80 °C at a rate of 10 °C min−1 under nitrogen atmosphere, (flow rate of 10 mL·min−1).

      Rheological analysis

    • Steady shear tests were carried out in a DHR-III rheometer (TA-Instrument, Delaware, USA), at a temperature of 25 °C, in a shear rate range of 0.1 to 100 s−1, with rough parallel plate geometry. The results were adjusted to the (Ostwald-de-Waale) power law model, using the ARES software (TA-Instrument, Delaware, USA). The evaluation of the linear viscoelasticity range was carried out in all the samples by means of a strain sweep in a range of 0.1% to 100%.

      For oscillatory test It was carried out in a DHR-III rheometer (TA-Instrument, Delaware, USA), at room temperature and a geometry of rough parallel plate geometry 40 mm with 1,000 µm gap[28,29].

      Creep–Compliance tests were performed on the bigels following the methodology proposed by Ojeda-Serna et al[30]. Ten Pa of stress was applied, and the response registered by 180 s, after that, force was retired, and compliance registered. The obtained results were modelling using the Burgers model.

      Cox-Merz rule

    • The superimposition of the shear rate dependence of steady shear viscosity, and the frequency dependence of complex viscosity, η*(ω), at equal values of frequency and shear rate was carried out using the Cox–Merz rule[23]:

      η∗(w)=η(γ)│w=γ (1)

      Where η* is complex viscosity, w is the frequency, η is the viscosity, γ is the shear rate.

      This rule provides insight on the structure of materials. Deviation from Cox–Merz rule is an indication of structural heterogeneity in materials[31].

      Statistical analysis

    • Data analysis was performed using the ANOVA test and mean comparison tests using the Tukey method (p < 0.05). The determination of parameters of the model used was obtained by non-linear estimation, using the Levenberg-Marquadt method, using the Statistica 12 software (StatSoft, Tulsa, OK, USA).

    Results

      Texture analysis

    • Results of the texture analysis are shown in Table 1. Firmness was defined as the maximum value of force in a plot of force vs time (positive peak), while the cohesiveness, was defined as the maximum value of the force in the negative peak arising during the movement of the probe upwards[27], this value could be used as the main parameter pointer of sample adhesion ability[5].

      Table 1.  Firmness and cohesiveness values of the bigels obtained at the different `proportions of hydrocolloid/oleogel at different agitation rates and mixing time.

      Hydrocolloid/
      oleogel      		Agitation
      (rpm)      		Mixing
      time (min)      		Firmness
      (N)      		Cohesiveness
      (N)
      67:3311,20051.19 ± 0.10ab−0.66 ± 0.001ab
      67:3311,20071.48 ± 0.10ab−0.73 ± 0.030ab
      67:3315,60051.60 ± 0.03b−0.76 ± 0.010b
      67:3315,60071.55 ± 0.18ab−0.69 ± 0.120ab
      83:1711,20050.88 ± 0.04ª−0.43 ± 0.020ab
      83:1711,20070.92 ± 0.16ab−0.42 ± 0.090ª
      83:1715,60051.02 ± 0.12ab−0.48 ± 0.010ab
      83:1715,60071.03 ± 0.11ab−0.48 ± 0.040ab
      Data shown are the mean of three replicates ± standard deviation; different literals in the same column indicates statistical differences (p < 0.05, Tukey test).

      Thermal analysis

    • Results obtained by DSC analysis of bigels are showed in Table 2, the value of melting temperature was influenced only for the interaction of hydrogel/oleogel × mixing rate (p < 0.05), Highest melting point (Tm) (2.2 °C) was obtained at 67:33 hydrogel/oleogel ratio and 15,600 rpm.

      Table 2.  Calorimetric behavior (melting, crystallization) of the bigels obtained at the different proportions of hydrocolloid/oleogel at different agitation rates and mixing time.

      Hydrocolloid/
      oleogel      		Agitation
      (rpm)      		Mixing time
      (min)      		Melting point
      (Tm) (°C)      		Melting enthalphy
      (Kcal/mol)      		Crystallization point
      (°C)      		Crystallization enthalphy
      (Kcal/mol)
      67:3311,20050.9 ± 0.166.32 ± 17.43−20.1 ± 0.375.99 ± 15.41
      67:3311,20071.5 ± 0.2133.15 ± 19.15−16.6 ± 0.7128.06 ± 11.74
      67:3315,60051.9 ± 0.2141.85 ± 13.35−14.5 ± 0.2129.40 ± 12.30
      67:3315,60072.2 ± 0.6164.05 ± 17.25−13.8 ± 0.3143.70 ± 13.80
      83:1711,20051.5 ± 0.2192.70 ± 23.80−16.8 ± 0.1163.40 ± 18.90
      83:1711,20071.4 ± 0.1201.10 ± 28.40−17.0 ± 0.7174.80 ± 17.40
      83:1715,60050.9 ± 0.159.34 ± 15.75−18.3 ± 0.286.50 ± 15.81
      83:1715,60071.4 ± 0.3195.6 ± 11.00−15.7 ± 0.7151.25 ± 20.15
      Data shown are the mean of three replicates ± standard deviation; different literals in the same column indicates statistical differences (p < 0.05, Tukey test).

      Rheological behavior

      Creep-Compliance

    • The Burgers model use Maxwell and Kelvin elements to study viscoelastic properties of the material, in short, G0 is a measurement of the sample resistance to deformation occurring immediately during the deformation profile. G0 is instantaneously recovered once the stress is removed. G1 is the contribution of the retarded elastic region to the total compliance; η0 is the residual viscosity or viscous flow of the system after suffering deformation, and η1 is the internal viscosity.[31]

      J(t)=1Go+1G1[1−exp(−tG1η1)]+t/η0 (2)

      where G0 is the instantaneous elastic modulus of the Maxwell unit, G1 is the elastic modulus of Kelvin–Voigt. The dashpot of the Maxwell element represents the residual viscosity, η0, and the dashpot associated with Kelvin–Voigt is called the internal viscosity, η1. Parameters Go, G1. All parameters yielded values of r2 ≥ 0.94, which can be considered satisfactory (Table 3).

      Table 3.  Parameters of Burgers model of bigels at the different conditions of ratio hydrogel (H)/Oleogel (O), mixing rate, time of agitation.

      Ratio H/ORate (rpm)Time (min)Recovery (%)G0 Paμ0 Pa sG1 Paμ1 Pa sλ sR2
      67:3311,200547.6310.54978.558.8518.512.090.96
      67:3311,200748.066.65709.007.6215.692.060.94
      67:3315,600533.006.78919.138.7811.131.270.93
      67:3315,600760.0418.91852.747.7933.294.270.98
      83:1711,200548.415.03575.485.717.651.340.92
      83:1711,200776.1129.82581.346.4841.066.340.99
      83:1715,600567.3353.11577.186.6350.407.600.99
      83:1715,600766.0871.221402.5120.12174.168.650.99
      Data shown are the mean of three replicates ± standard deviation.
      Cox-Mertz

    • Superposition of data from shear viscosity and complex viscosity was observed over the wide shear rate and/or frequency range for all samples, as illustrated in Fig. 1.

      Figure 1. 

      Cox–Merz rule applied to bigels obtained with oleogels and hydrogels at all experimental condition used in the experimental work.

    Discussion

      Texture analysis

    • When the concentration of hydrogel increased from 63% to 87%, the firmness of bigel decreased (p < 0.05), major differences were observed between samples at low rate of agitation and high time of mixing, on the other hand, lower differences were observed for low rate of agitation and low time of mixing.

      This behavior indicates that not only plasticing effect of surfactant molecules affect the firmness[27], but it is also important to evaluate process parameters involved in the obtention of bigels. Usually, surfactant increase Van der Waals interactions and hydrogen bonds between polymer strands, increasing the firmness of the gels. However, high rate of mixing and high time of mixing could be introduce air bubbles into the system, the presence of the new material into the system (air) affects the stability of the system, producing instabilities that diminish the firmness of the bigels[32].

      Higher cohesiveness is an indication of the presence of tridimensional structure and then more consistency of the gel. Thus, in general, the higher presence of oleogel into the bigel (33% w/w) showed high cohesiveness. This could be attributed to the formation of an emulsion-like semisolid system with a more complex three-dimensional structure with droplets of oleogel dispersed in the hydrogel, more experiments are needed to probe the influence of the morphology in the texture behavior. Similar results have been reported by several authors[5,33], they observed higher hardness of emulsions and bigels when increasing the oil phase.

      Thermal analysis

    • An increase in the melting temperature at higher amount of oleogel was reported by Singh et al.[34] for bigels made with sorbitan monostearate-sesame oil organogel and carbopol 934 hydrogel, however, they explained that higher proportion of oleogel increased the solid fraction and then higher temperatures were necessary to melt the bigel, however, they did not evaluate the influence of mixing rate, mixing rate apply a shear to the sample, results obtained indicate that high shear and high presence of oleogel produce more ordered systems than needing higher temperatures for melting.

      Also, the melting enthalphy was influenced by the hydrogels/oleogel × mixing rate (p < 0.05), however, main effects (ratio hydrogel/oleogel, mixing rate) and the interaction of hydrogels/oleogel × mixing rate × mixing time showed statistical significance (p < 0.05), thus, the operational variables (mixing time, mixing rate) and the ratio of hydrogel/oleogel contribute to obtain specific melting temperature. The highest enthalpy of fusion was observed at the condition of lower amount of oleogel, lower stirring speed and longer stirring time. On the other hand, the lowest enthalpy of fusion was observed at the condition of higher amount of oleogel, lower stirring speed and lower mixing time. The higher value of melting entalphy could be related with the closer packing of the dispersed phase, related with the lower amount of oleogel, lower stirring rate and higher mixing time, that modify the size of the lipid particle[35], more experiments on particle size and morphology would help to clarifying this point.

      In the present experiment, exothermic peak related with the crystallization was observed at the range of −13 to −20 °C (broader range than endothermic behavior), at the observed temperatures, the crystallization peak could be related with the canola oil in oleogel phase, this behavior is opposite of that reported by Singh et al.[10], who claim that the crystallization behavior of bigels is related with the presence of the gellators, however, in the present experiment, the crystallization of Myverol was around 52 °C, thus, the crystallization behavior was related with the presence of the canola oil into the bigels. The obtained results are in agreement with the reports by Contreras-Ramirez et al.[36], who reported for gelled emulsions made with canola oil, a crystallization temperature of −20 °C, like that found in the present work. There is a significant difference between melting and crystallization temperatures, however, the values of melting and crystallization temperatures do not show significant changes, this behavior has been reported by Habibi et al.[37].

      Rheological behavior

    • All Parameters G0, G1, ƞ0, and ƞ1 showed a strong dependence of process parameters (hydrogel/oleogel ratio, mixing rate and mixing time; (p < 0.05), also, all statistical interactions were significative (p < 0.05). Higher G0 value was observed at lower amount of oleogel (17%) into the bigels and higher mixing rate. When more mixing time was used, independent of the proportion of hydrogel/oleogel, it obtained the highest value of G0, this behavior indicates lower deformation of the sample at the same imposed stress. Similar behavior was observed for G1. It is interesting that the contribution of G elements of Kelvin Voight and Maxwell, indicate that ratio G1/G0 was similar or lower than 1, indicating prevalence of the Maxwell element over the Kelvin Voight element, like the gelled emulsions reported by Ojeda-Serna et al.[30]. Major prevalence of the Maxwell model element was observed at lower amounts of oleogel, however statistical interaction (mixing rate and mixing time) is present indicating a complex system.

      Higher values of recovery were observed with bigels with lower amount of oleogel (17%), also a strong statistical interaction was present (mixing time and mixing rate). Highest recovery (76%) was observed at lower proportions of oleogel, higher mixing time and lower mixing rate. On the other hand, lowest recovery (33%), was observed at high proportions of oleogel, high mixing rate and low time of mixing. High viscosity parameter was observed with bigels with high proportion of oleogel. The contribution of the viscous element to the compliance of bigels was higher than the contribution of dashpot elements, thus, the bigels obtained show a high viscosity with a lower structuration (dashpot), opposite to the behavior observed in organogels and gelled emulsions[26] (Table 3).

      The empirical Cox–Merz superposition of steady-shear viscosity and oscillatory shear is consistent with topological entanglement interactions of individual species[38]. Also, the frequency sweep tests showed a significant frequency dependence of both moduli (Fig. 1) and the decrease in G0 with increasing amplitude of oscillation started at lower stresses, this behavior is usual in gels, especially when is they present weak topological interactions of polymer chains (founded into the hydrogel). In this sense, the gels are more sensitive to strain than solutions or dispersions.

      The deviations from the Cox–Merz rule has been related to the specific polymeric and the entanglements founded in system with gel-like behavior[39]. Also, Vernon-Carter et al.[40] claim that the Cox-Merz rule usually fits well in a system showing a fluid-like or solid-like behavior.

      In the field of bigels no reports about the use of Cox-Mertz rule have been carried out, however, Kwak et al.[41] use the Cox-Mertz rule to probe topical cream and ointments, finding that, these systems do not obey the rule. A system that fits the Cox-Merz rule indicated that the strain magnitude applied to the material does not provoke a structural decay.

      Thus, the nature of the relationship displayed in Fig. 1 for the bigels were consistent with the Cox-Merz rule and indicates that these formulations are polymer dispersions. As expected, at high values of shear rate, the structure of the material is breaking, showing deviations from Cox-Merz rule at values higher than 30 s−1 of shear rate, however, at lower values of shear rate the Cox-Merz rule described very well the obtained bigels. Few examples of of emulsions that have been modelled by the Cox-Merz rule, have been found in the literature, Murillo-Martínez et al.[42] reported a multiple emulsion that fits very well to the Cox-Merz rule, and Medina-Torres et al.[43] indicate that highly structured systems did not obey this rule, opposite to the behavior of a polymeric dispersion, also, the use of master curves or time-temperature superposition approaches[44] could improve the knowledge about the bigels mechanical behavior.

    Conclusions

    • Mixing time, mixing rate and rate of hydrogel/oleogel influence mechanical properties of bigels made with canola oil, myverol, guar gum and water. Better mechanical properties were obtained at lower mixing time, higher mixing rate and low proportion of oleogel in the bigels. The Cox-Merz rule described the complex viscosity and shear viscosity at low frequencies and shear rate in bigels.

    Acknowledgments

    • JDMM acknowledges Conacyt for their scholarship.

      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 Nanjing 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 (1)  Table (3) References (44)
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    Cite this article
    Mata-Mota JD, Gallegos-Infante JA, Pérez-Martínez JD, Rocha-Guzmán NE, González-Laredo RF. 2023. Effect of hydrogel/oleogel ratio, speed and time of mixing, on the mechanical properties of bigel materials and the application of Cox-Merz rule. Food Materials Research 3:24 doi: 10.48130/FMR-2023-0024
    Mata-Mota JD, Gallegos-Infante JA, Pérez-Martínez JD, Rocha-Guzmán NE, González-Laredo RF. 2023. Effect of hydrogel/oleogel ratio, speed and time of mixing, on the mechanical properties of bigel materials and the application of Cox-Merz rule. Food Materials Research 3:24 doi: 10.48130/FMR-2023-0024
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  • Introduction
  • Materials and methods

  • Bigel preparation

  • Texture analysis

  • Thermal analysis

  • Rheological analysis

  • Cox-Merz rule

  • Statistical analysis

  • Results

  • Texture analysis

  • Thermal analysis

  • Rheological behavior

  • Creep-Compliance
  • Cox-Mertz
  • Discussion

  • Texture analysis

  • Thermal analysis

  • Rheological behavior

  • Conclusions
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
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