Enzyme kinetic approach for mechanistic insight and predictions of in vivo starch digestibility and the glycaemic index of foods.

BACKGROUND: Starch is a principal dietary source of digestible carbohydrate and energy. Glycaemic and insulinaemic responses to foods containing starch vary considerably and glucose responses to starchy foods are often described by the glycaemic index (GI) and/or glycaemic load (GL). Low GI/GL foods are beneficial in the management of cardiometabolic disorders (e.g., type 2 diabetes, cardiovascular disease). Differences in rates and extents of digestion of starch-containing foods will affect postprandial glycaemia. SCOPE AND APPROACH: Amylolysis kinetics are influenced by structural properties of the food matrix and of starch itself. Native (raw) semi-crystalline starch is digested slowly but hydrothermal processing (cooking) gelatinises the starch and greatly increases its digestibility. In plants, starch granules are contained within cells and intact cell walls can limit accessibility of water and digestive enzymes hindering gelatinisation and digestibility. In vitro studies of starch digestion by α-amylase model early stages in digestion and can suggest likely rates of digestion in vivo and expected glycaemic responses. Reports that metabolic responses to dietary starch are influenced by α-amylase gene copy number, heightens interest in amylolysis. KEY FINDINGS AND CONCLUSIONS: This review shows how enzyme kinetic strategies can provide explanations for differences in digestion rate of different starchy foods. Michaelis-Menten and Log of Slope analyses provide kinetic parameters (e.g., K m and k cat /K m ) for evaluating catalytic efficiency and ease of digestibility of starch by α-amylase. Suitable kinetic methods maximise the information that can be obtained from in vitro work for predictions of starch digestion and glycaemic responses in vivo.

A single base change at exon of Wx-A1 caused gene inactivation and starch properties modified in a wheat EMS mutant line.

BACKGROUND: Wheat is an essential source of starch. The GBSS or waxy genes are responsible for synthesizing amylose in cereals. The present study identified a novel Wx-A1 null mutant line from an ethyl methanesulfonate (EMS)-mutagenized population of common wheat cv. SM126 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel analyses. RESULTS: The alignment of the Wx-A1 gene sequences from the mutant and parental SM126 lines showed only one single nucleotide polymorphism causing the appearance of a premature stop codon and Wx-A1 inactivation. The lack of Wx-A1 protein resulted in decreased amylose, total starch and resistant starch. The starch morphology assessment revealed that starch from mutant seeds was more wrinkled, increasing its susceptibility to digestion. Regarding the starch thermodynamic properties, the gelatinization temperature was remarkably reduced in the mutant compared to parental line SM126. The digestibility of native, gelatinized, and retrograded starches was analyzed for mutant M4-627 and the parental SM126 line. In the M4-627 line, rapidly digestible starch contents were increased, whereas resistant starch was decreased in the three types of starch. CONCLUSION: Waxy protein is essential for starch synthesis. The thermodynamic characteristics were decreased in the Wx-A1 mutant line. The digestibility properties of starch were also affected. Therefore, the partial waxy mutant M3-627 might play a significant role in food improvement. Furthermore, it might also be used to produce high-quality noodles. © 2021 Society of Chemical Industry.

Analysis of starch structure and functional properties of tetraploid wheat (Triticum turgidum L.) with differing waxy protein composition.

BACKGROUND: An increased demand for food has mirrored the increasing global population. Obesity and diabetes are two disorders induced by poor eating choices. Consequently, there is an urgent need to develop modified foods that can ameliorate such illnesses. The objective of this study was to explore the effect of Waxy genes on the structural and functional properties of starch, with the aim of improving food quality. Wild-type tetraploid wheat was compared with three mutants with different Waxy gene combinations. RESULTS: The proportion of B-type granules was higher in the mutants than in the wild-type (Wx-AB), and there were significant changes in the starch granule size, number, and phenotype in the Wx free mutant (Wx-ab). The lowest branch chain length was observed in Wx-ab, whereas Wx-AB had the highest branch chain length of DP ≥ 37. Wx-ab had the highest degree of crystallinity. The crystallinity trend followed the order Wx-ab>Wx-Ab>Wx-aB>Wx-AB. The amount of slowly digestible starch (SDS) was higher in native, gelatinized, and retrograded starch in the mutant. The amount of retrograded starch was closer to gelatinized starch than to native starch. CONCLUSION: Waxy proteins make a substantial contribution to starch structure. A lack of waxy proteins reduced the unit chains markedly compared with the control. Waxy proteins significantly affected the smaller and longer chains of starch. The lines with differing waxy composition had different effects on food digestion. The Wx-AB in native starch and Wx-Ab in gelatinized starch can control obesity and diabetes by slow-digesting carbohydrates and high resistance to digestion. © 2022 Society of Chemical Industry.

Expression and characterization of 1,4-α-glucan branching enzyme from Microvirga sp. MC18 and its application in the preparation of slowly digestible starch.

Nutraceuticals containing modified starch with increased content of slowly-digestible starch (SDS) may reduce the prevalence of obesity, diabetes and cardiovascular diseases due to its slow digestion rate. Enzymatic methods for the preparation of modified starch have attracted increasing attention because of their low environmental impact, safety and specificity. In this study, the efficient glucan branching enzyme McGBE from Microvirga sp. MC18 was identified, and its relevant properties as well as its potential for industrial starch modification were evaluated. The purified McGBE exhibited the highest specificity for potato starch, with a maximal specific activity of 791.21 U/mg. A time-dependent increase in the content of α-1,6 linkages from 3.0 to 6.0% was observed in McGBE-modified potato starch. The proportion of shorter chains (degree of polymerization, DP < 13) increased from 29.2 to 63.29% after McGBE treatment, accompanied by a reduction of the medium length chains (DP 13-24) from 52.30 to 35.99% and longer chains (DP > 25) from 18.51 to 0.72%. The reduction of the storage modulus (G') and retrogradation enthalpy (ΔHr) of potato starch with increasing treatment time demonstrated that McGBE could inhibit the short- and long-term retrogradation of starch. Under the optimal conditions, the SDS content of McGBE-modified potato starch increased by 65.8% compared to native potato starch. These results suggest that McGBE has great application potential for the preparation of modified starch with higher SDS content that is resistant to retrogradation.

Starch-guest inclusion complexes: Formation, structure, and enzymatic digestion.

Starch/amylose-guest inclusion complexes, a class of supramolecular host-guest assemblies, are of critical importance in the processing, preservation, digestion, nutrients/energy uptake, and health outcomes of starch-containing foods. Particularly, the formation of inclusion complex has been suggested to lower the rate and extent of enzymatic digestion of starch and starch-containing foods. Compared with rapidly digestible starch, starch inclusion complex may fall into the category of slowly digestible starch, providing sustained glucose release and maintaining glucose homeostasis. Therefore, the ability of starch-guest inclusion complex to alter the digestive behavior of energy-dense starchy foods has been of interest to many researchers and has the potential to be developed and formulated into functional foods. In this article, we provide a comprehensive and critical review on the current knowledge of the in vitro and in vivo enzymatic digestion of starch-guest inclusion complexes, by emphasizing the structure-digestibility relationship. We examine the preparation methods employed, crystalline structures obtained, and physicochemical properties characterized in previous reports, which all have implications on the digestive behavior reported on the starch-guest inclusion complexes. In addition, we give suggestions on future research to elucidate the digestive properties of starch-guest inclusion complexes and to develop functional structures based on these complexes for use in foods and nutrition.

Carbohydrates designed with different digestion rates modulate gastric emptying response in rats.

We sought to determine whether design of carbohydrate-based microspheres to have different digestion rates, while retaining the same material properties, could modulate gastric emptying through the ileal brake. Microspheres made to have three slow digestion rates and a rapidly digested starch analogue (maltodextrin) were administrated to rats by gavage and starch contents in the stomach, proximal and distal small intestine, and caecum were measured 2 h post-gavage. A stepwise increase in the amount of starch retained in the stomach was found for microspheres with incrementally slower rates of digestion. Postprandial glycaemic and insulinaemic responses were incrementally lower for the different microspheres than for the rapidly digestible control. A second-meal effect was observed for slowly digestible starch (SDS) microspheres compared to glucose. Thus, dietary slowly digestible carbohydrates were designed to elicit incremental significant changes in gastric emptying, glycaemic and insulinaemic responses, and they may be a means to trigger the ileal brake.

Starch digestibility: past, present, and future.

In the last century, starch present in foods was considered to be completely digested. However, during the 1980s, studies on starch digestion started to show that besides digestible starch, which could be rapidly or slowly hydrolysed, there was a variable fraction that resisted hydrolysis by digestive enzymes. That fraction was named resistant starch (RS) and it encompasses those forms of starch that are not accessible to human digestive enzymes but can be fermented by the colonic microbiota, producing short-chain fatty acids. RS has been classified into five types, depending on the mechanism governing its resistance to enzymatic hydrolysis. Early research on RS was focused on the methods to determine its content in foods and its physiological effects, including fermentability in the large intestine. Later on, due to the interest of the food industry, methods to increase the RS content of isolated starches were developed. Nowadays, the influence of RS on the gut microbiota is a relevant research topic owing to its potential health-related benefits. This review summarizes over 30 years of investigation on starch digestibility, its relationship with human health, the methods to produce RS and its impact on the microbiome. © 2018 Society of Chemical Industry.

Starch digestion kinetics and mechanisms of hydrolysing enzymes in growing pigs fed processed and native cereal-based diets.

This study aimed to examine in vivo starch digestion kinetics and to unravel the mechanisms of starch hydrolysing enzymes. Ninety pigs (23 (sd 2·1) kg body weight) were assigned to one of nine treatments in a 3×3 factorial arrangement, with starch source (barley, maize, high-amylose (HA) maize) and form (isolated, within cereal matrix, extruded) as factors. We determined starch digestion coefficients (DC), starch breakdown products and digesta retention times in four small-intestinal segments (SI1-4). Starch digestion in SI2 of pigs fed barley and maize, exceeded starch digestion of pigs fed HA maize by 0·20-0·33 DC units (P<0·01). In SI3-4, barley starch were completely digested, whereas the cereal matrix of maize hampered digestion and generated 16 % resistant starch in the small intestine (P<0·001). Extrusion increased the DC of maize and HA maize starch throughout the small intestine but not that of barley (P<0·05). Up to 25 % of starch residuals in the proximal small intestine of pigs was present as glucose and soluble α(1-4) maltodextrins. The high abundance of glucose, maltose and maltotriose in the proximal small intestine indicates activity of brush-border enzymes in the intestinal lumen, which is exceeded by α-amylase activity. Furthermore, we found that in vivo starch digestion exceeded our in vitro predictions for rapidly digested starch, which indicates that the role of the stomach on starch digestion is currently underestimated. Consequently, in vivo glucose release of slowly digestible starch is less gradual than expected, which challenges the prediction quality of the in vitro assay.

High amylose starch consumption induces obesity in Drosophila melanogaster and metformin partially prevents accumulation of storage lipids and shortens lifespan of the insects.

There are very few studies that have directly analyzed the effects of dietary intake of slowly digestible starches on metabolic parameters of animals. The present study examined the effects of slowly digestible starch with high amylose content (referred also as amylose starch) either alone, or in combination with metformin on the development, lifespan, and levels of glucose and storage lipids in the fruit fly Drosophila melanogaster. Consumption of amylose starch in concentrations 0.25-10% did not affect D. melanogaster development, whereas 20% starch delayed pupation and reduced the number of larvae that reached the pupal stage. Starch levels in larval food, but not in adult food, determined levels of triacylglycerides in eight-day-old adult flies. Rearing on diet with 20% starch led to shorter lifespan and a higher content of triacylglycerides in the bodies of adult flies as compared with the same parameters in flies fed on 4% starch diet. Food supplementation with 10mM metformin partly attenuated the negative effects of high starch concentrations on larval pupation and decreased triacylglyceride levels in adult flies fed on 20% starch. Long-term consumption of diets supplemented with metformin and starch decreased lifespan of the insects, compared with the diet supplemented with starch only. The data show that in Drosophila high starch consumption may induce a fat fly phenotype and metformin may partially prevent it.

The nutritional property of endosperm starch and its contribution to the health benefits of whole grain foods.

Purported health benefits of whole grain foods in lowering risk of obesity, type 2 diabetes, cardiovascular disease, and cancer are supported by epidemiological studies and scientific researches. Bioactive components including dietary fibers, phytochemicals, and various micronutrients present in the bran and germ are commonly considered as the basis for such benefits. Endosperm starch, as the major constituent of whole grains providing glucose to the body, has been less investigated regarding its nutritional property and contribution to the value of whole grain foods. Nutritional quality of starch is associated with its rate of digestion and glucose absorption. In whole grain foods, starch digestion and glucose delivery may vary depending on the form in which the food is delivered, some with starch being rapidly and others slowly digested. Furthermore, there are other inherent factors in whole grain products, such as phenolic compounds and dietary fibers, that may moderate glycemic profiles. A good understanding of the nutritional properties of whole grain starch is important to the development of food processing technologies to maximize their health benefits.

Slowly digestible starch--a review.

The link between carbohydrate intake and health is becoming increasingly important for consumers, particularly in the areas of glycemic index (GI) and extended energy-releasing starches. From a physiological point of view, slowly digestible starch (SDS) delivers a slow and sustained release of blood glucose along with the benefits resulting from low glycemic and insulinemic response. SDS has been implicated in several health problems, including diabetes, obesity, and cardiovascular diseases (metabolic syndromes). It may also have commercial potential as a novel functional ingredient in a variety of fields, such as nutrition, medicine, and agriculture. The present review assesses this form of digestion by analyzing methods to prepare and evaluate SDS, and factors affecting its transformation, its health benefits, and its applications.

Recrystallization of starches by hydrothermal treatment: digestibility, structural, and physicochemical properties.

Gelatinized starches were recrystallized under hydrothermal treatment and their properties were characterized by X-ray diffractometry, solid-state (13)C cross-polarization and magic-angle spinning nuclear magnetic resonance, differential scanning calorimetry, gel-permeation chromatography, high-performance anion-exchange chromatography using pulsed amperomeric detection, high-performance size-exclusion chromatography with attached multiangle laser light scattering and refractive index detectors, and digestibility analysis. Amylopectin molecules of hylon (V, VII) and water yam starch contained long side-chains with high proportion of fb1 and fb2. Under hydrothermal treatment, the double helix proportion and relative crystallinity significantly increased and reached maxima of water yam (48.7 and 28.2 %, respectively). Except water yam starch, X-ray diffraction pattern of all starches exhibited the evidence of type 2 amylose-lipid complex. Besides, under DSC measurement, potato and hylon starches showed the endotherm of amylose-amylose interaction. The hydrothermal treatment caused the recrystallization resulting in the decrease of RDS, especially in case of hylon and water yam starch. HTT water yam contained highest SDS (48.3 %) and HTT hylon VII contained highest RS (44.5 %). The relationship between structure and digestibility was observed, in which, high amylose content and specific structures of amylopectin molecule were necessary for the production of RS and/or SDS of hydrothermally treated starches.

Effect of ethanol addition on the physicochemical, structural and in vitro digestive properties of Tartary buckwheat starch-quercetin/rutin complexes.

The effects of ethanol on the physicochemical, structural and in vitro digestive properties of Tartary buckwheat starch-quercetin/rutin complexes (e-TBSQ and e-TBSR) were investigated. Ethanol restricted the gelatinization of Tartary buckwheat starch (TBS), which resulted an increase in ∆H, G' and G" as well as a decrease in apparent viscosity of e-TBSQ and e-TBSR. The particle size, scanning electron microscopy and X-ray diffraction results showed that ethanol influenced the morphological structure of TBS granules and the starch crystalline structure in e-TBSQ and e-TBSR changed from B-type to V-type when the ethanol concentration was 25%. Saturation transfer difference-nuclear magnetic resonance results revealed that ethanol weakened the binding ability of quercetin/rutin to TBS in e-TBSQ and e-TBSR, leading to a change in the binding site on the quercetin structural unit. The residual ungelatinized TBS granules in e-TBSQ and e-TBSR induced a high slowly digestible starch content, and thus displayed a "resistant-to-digestion".

Effects of color protection and enzymatic hydrolysis on the microstructure, digestibility, solubility and swelling degree of chestnut flour.

Chestnuts, despite their nutritional value, pose challenges in starch processing, digestion, and absorption. This study employed various color-fixing formulations and processing methods to simulate the in vitro digestion of both untreated and enzymatically hydrolyzed chestnut flour. Changes in starch properties, digestion characteristics, and estimated glycemic index (eGI) were analyzed to understand how enzymatic hydrolysis affects chestnut flour properties. The results showed that the browning of chestnut flour was the least when the mass ratio of vitamin C, citric acid, and EDTA-Na2 was 9:1:0.3. Following treatment with pullulanase and glucoamylase, the content of rapidly digestible starch decreased to 10 %, while the content of slowly digestible starch and resistant starch increased to 62 % and 27 %, respectively. The eGI value of chestnut flour after enzymatic hydrolysis increased to 61.85-65.14, the hydrolysis rate was 78.37 %-89.20 %, the water holding capacity was 5.3-8.6, the solubility was 51.33 %-58.33 %, and the swelling degree decreased to 2.21-3.33 mL/g.

Novel one-step method to construct gellan gum-zein core-shell structured starch beads for regulating starch digestion.

A simple and efficient one-step method combining ion crosslinking and antisolvent exchange has been developed to construct gellan gum/corn starch@zein (GG/CS@Z) core-shell structured beads. This novel approach aims to reduce the digestibility and digestion rate of starch. The GG/CS@Z beads were comprehensively characterized using scanning electron microscopy (SEM), confocal laser scanning microscope (CLSM), differential scanning calorimetry (DSC), swelling power experiments and in vitro simulated digestion tests, respectively. SEM and CLSM analyses unequivocally confirmed the successful construction of the core-shell structure in GG/CS@Z beads. The encapsulation of starch within the core-shell structure effectively restricted its swelling and gelatinization by inhibiting water contact. Notably, compared to native corn starch, the GG/CS@Z5 beads exhibited significantly enhanced contents of slowly digestible starch (SDS) and resistant starch (RS), reaching 34.07 % and 26.86 %, respectively. These findings demonstrate the potential of GG/CS@Z core-shell structured beads as functional food ingredients for individuals with cardiovascular diseases and diabetes.

Interaction between starch and dietary compounds: New findings and perspectives to produce functional foods.

Due to the increased prevalence of overweight, obesity, diabetes, colon cancer, cardiovascular diseases, and metabolic syndrome, dietary approaches to reduce starch digestion and regulate glucose homeostasis have gained attention. Starch is a polysaccharide in most daily food consumed as bakery products, snacks, breakfast cereals, and pasta, which are often vilified. However, it is also present in beans, lentils, and oatmeal, which are considered healthy food products. The difference relays on the food matrix and the thermal process that can produce interactions between starch and dietary compounds (protein, lipid, non-starch polysaccharide, and bioactive compounds) or among starch chains (retrogradation). Such interactions produce structural changes so the digestive enzymes cannot hydrolyze them; additionally, the physical barrier of some macromolecules (proteins, hydrocolloids) restricts starch gelatinization and accessibility of the digestive enzymes to hydrolyze the starch. The interactions mentioned above and the use of some macromolecules as physical barriers could be explored as a pathway to develop functional foods. This review analyzes the interactions between starch and dietary compounds influenced by the processing of some food matrices to better understand their potential for developing functional foods.

Biochemical characteristics and potential application of a thermostable starch branching enzyme from Bacillus licheniformis.

Slowly digestible starch (SDS) has attracted increasing attention for its function of preventing metabolic diseases. Based on transglycosylation, starch branching enzymes (1,4-α-glucan branching enzymes, GBEs, EC 2.4.1.18) can be used to regulate the digestibility of starch. In this study, a GBE gene from Bacillus licheniformis (bl-GBE) was cloned, expressed, purified, and characterized. Sequence analysis and structural modeling showed that bl-GBE belong to the glycoside hydrolase 13 (GH13) family, with which its active site residues were conserved. The bl-GBE was highly active at 80 °C and a pH range of 7.5-9.0, and retained 90% of enzyme activity at 70 °C for 16 h. bl-GBE also showed high substrate specificity (80.88 U/mg) on potato starch. The stability and the changes of the secondary structure of bl-GBE at different temperature were determined by circular dichroism (CD) spectroscopy. The CD data showed a loss of 20% of the enzyme activity at high temperatures (80 °C), due to the decreased content of the α -helix in the secondary structure. Furthermore, potato starch treated with bl-GBE (300 U/g starch) showed remarkable increase in stability, solubility, and significant reduction viscosity. Meanwhile, the slowly digestible starch content of bl-GBE modified potato starch increased by 53.03% compared with native potato starch. Our results demonstrated the potential applications of thermophilic bl-GBE in food industries.

Yellow Mealworm (Tenebrio molitor) Powder Promotes a High Bioaccessible Protein Fraction and Low Glycaemic Index in Biscuits.

Traditional biscuits are considered products with poor nutritional value because of their large share of rapidly digested starch, which results in an elevated glycaemic index. This paper explores the improvement of the nutritional value of biscuits by adding yellow mealworm (Tenebrio molitor) powder. Four biscuit recipes containing 0%(R1), 10%(R2), 15%(R3), and 20%(R4) of yellow mealworm powder were prepared and subjected to sensorial analysis. The R3 biscuits were selected for further investigation, as they had the highest acceptability. Compared to the reference R1, the R3 biscuits showed an improved nutritional profile in terms of protein, fat, ash, minerals, fibres, essential amino acids, and unsaturated fatty acids, and lower amounts of carbohydrates and 5-hydroxymethylfurfural. The in vitro protein digestibility in R3 improved 1.12-fold compared to R1. No significant difference was found between the digestibility of the lipids released from R1 and R3. A higher fraction of slowly digestible starch was present in R3 compared to R1. The starch digestibility and estimated glycaemic index were 72.96% and 79.56% in R3, which can be compared to 78.79% and 90.14%, respectively, in R1. Due to their enhanced nutritional profile, higher bioaccessible protein fraction, and lower glycaemic index, yellow mealworm powder biscuits can be considered a more nutritious alternative to traditional biscuits.

Effect of the Pasta Making Process on Slowly Digestible Starch Content.

The rate at which starch is digested in the human intestine elicits different glycemic responses and reflects the glycemic index (GI) of foods. In vitro measurement of starch digestibility can reflect the GI of food. Differences in starch digestibility among four durum wheat pasta samples, couscous, and bread were evaluated to better describe the role of the pasta making process in affecting starch digestibility. Statistical differences in RDS (rapidly digestible starch), SDS (slowly digestible starch), and RS (resistant starch) of products were found (p < 0.05). As expected, pasta samples showed the highest value of SDS/av starch compared to couscous and bread. Fusilli and cavatelli samples presented the highest SDS/av starch ratio (55.80 ± 3.06% and 53.91 ± 3.50%, respectively), then came spaghetti 49.39 ± 2.83% and penne 45.93 ± 1.19%, while couscous presented the lowest value of SDS/av starch (2.64 ± 0.50%), followed by bread (11.78 ± 2.63%). Our study confirmed that the pasta making process efficiently mediates an increase in SDS/Av starch content, which has been specifically quantified above 40%, therefore strongly related to a lowered glycemic response in vivo. Our results strengthened the concept that pasta is a good source of SDS, which makes it useful for glycemic control.

Genotype-by-environment interactions for starch, mineral, and agronomic traits in pearl millet hybrids evaluated across five locations in West Africa.

Introduction: Pearl millet is a staple cereal grown in the harshest environments of arid and semi-arid regions of Asia and sub-Saharan Africa. It is the primary source of calories for millions of people in these regions because it has better adaptation to harsh environmental conditions and better nutritional traits than many other cereals. By screening the pearl millet inbred germplasm association panel (PMiGAP), we earlier reported the best genotypes with the highest concentration of slowly digestible and resistant starch in their grains. Methods: In the current study, we tested these 20 top-performing pearl millet hybrids, identified based on starch data, in a randomised block design with three replications at five locations in West Africa, viz. Sadore and Konni (Niger), Bambey (Senegal), Kano (Nigeria), and Bawku (Ghana). Phenotypic variability was assessed for agronomic traits and mineral traits (Fe and Zn). Results and discussion: Analysis of variance demonstrated significant genotypic, environmental, and GEI effects among five testing environments for agronomic traits (days to 50% flowering, panicle length, and grain yield), starch traits (rapidly digestible starch, slowly digestible starch, resistant starch, and total starch), and mineral trait (iron and zinc). Starch traits, such as rapidly digestible starch (RDS) and slowly digestible starch (SDS), showed nonsignificant genotypic and environmental interactions but high heritability, indicating the lower environmental influence on these traits in the genotype × testing environments. Genotype stability and mean performance across all the traits were estimated by calculating the multi-trait stability index (MTSI), which showed that genotypes G3 (ICMX207070), G8 (ICMX207160), and G13 (ICMX207184) were the best performing and most stable among the five test environments.