Formation mechanism of quinoa protein hydrolysate-EGCG complexes at different pH conditions and its effect on the protein hydrolysate-lipid co-oxidation in emulsions.

This study aimed to investigate the interaction, structure, antioxidant, and emulsification properties of quinoa protein hydrolysate (QPH) complexes formed with (-)-epigallocatechin gallate (EGCG) at pH 3.0 and 7.0. Additionally, the effect of pH conditions and EGCG complexation on protein hydrolysate-lipid co-oxidation in QPH emulsions was explored. The results indicated that QPH primarily interacted with EGCG through hydrophobic interactions and hydrogen bonds. This interaction led to alterations in the secondary structure of QPH, as well as a decrease in surface hydrophobicity and free SH content. Notably, the binding affinity between QPH and EGCG was observed to be higher at pH 7.0 compared to pH 3.0. Consequently, QPH-EGCG complexes exhibited more significant enhancement in antioxidant and emulsification properties at pH 7.0 than pH 3.0. The pH level also influenced the droplet size, ζ-potential, and interfacial composition of emulsions formed by QPH and QPH-EGCG complexes. Compared to QPH stabilized emulsions, QPH-EGCG stabilized emulsions were more capable of mitigating destabilization during storage and displayed fewer lipid oxidation products, carbonyl generation, and sulfhydryl groups and fluorescence loss, which implied better oxidative stability of the emulsions. Furthermore, the QPH-EGCG complexes formed at pH 7.0 exhibited better inhibition of protein hydrolysate-lipid co-oxidation. Overall, these findings provide valuable insights into the potential application of QPH and its complexes with EGCG in food processing systems.

Encapsulation of chrysin and rutin using self-assembled nanoparticles of debranched quinoa, maize, and waxy maize starches.

Chrysin and rutin are natural polyphenols with multifaceted biological activities but their applications face challenges in bioavailability. Encapsulation using starch nanoparticles (SNPs) presents a promising approach to overcome the limitations. In this study, chrysin and rutin were encapsulated into self-assembled SNPs derived from quinoa (Q), maize (M), and waxy maize (WM) starches using enzyme-hydrolysis. Encapsulation efficiencies ranged from 74.3 % to 79.1 %, with QSNPs showing superior performance. Simulated in vitro digestion revealed sustained release and higher antioxidant activity in QSNPs compared to MSNPs and WMSNPs. Variations in encapsulation properties among SNPs from different sources were attributed to the differences in the structural properties of the starches. The encapsulated SNPs exhibited excellent stability, retaining over 90 % of chrysin and 85 % of rutin after 15 days of storage. These findings underscore the potential of SNP encapsulation to enhance the functionalities of chrysin and rutin, facilitating the development of fortified functional foods with enhanced bioavailability and health benefits.

Physicochemical and functional properties of short-chain fatty acid starch modified with different acyl groups and levels of modification.

Rice and quinoa starches are modified with short-chain fatty acids (SCFA) with different SCFA acyl chain lengths and levels of modification. This work is aimed to investigate the impact of modifying rice and quinoa starches with short-chain fatty acids (SCFAs) on various physicochemical properties, including particle size, protein and amylose content, thermal behavior, pasting characteristics, and in vitro digestibility. Both native and SCFA-starches showed comparable particle sizes, with rice starches ranging from 1.58 to 2.22 µm and quinoa starches from 5.18 to 5.72 µm. SCFA modification led to lower protein content in both rice (0.218-0.255 %) and quinoa starches (0.537-0.619 %) compared to their native counterparts. Esterification led to the reduction of gelatinization and pasting temperatures as well as the hardness of the paste of SCFA-starches were reduced while paste clarity increased. The highest level of modification in SCFA-starch was associated with the highest amount of resistant starch fraction. Principal component analysis revealed that modification levels exerted a greater influence on starch properties than the types of SCFA used (acetyl, propionyl, and butyryl). These findings is importance in considering the degree of substitution or level of modification when tailoring starch properties through SCFA modification, with implications for various applications in food applications.

Fermentation enrichment, structural characterization and immunostimulatory effects of ß-glucan from Quinoa.

We developed an efficient mixed-strain co-fermentation method to increase the yield of quinoa ß-glucan (Q+). Using a 1:1 mass ratio of highly active dry yeast and Streptococcus thermophilus, solid-to-liquid ratio of 1:12 (g/mL), inoculum size of 3.8 % (mass fraction), fermentation at 32 °C for 27 h, we achieved the highest ß-glucan yield of (11.13 ± 0.80)%, representing remarkable 100.18 % increase in yield compared to quinoa ß-glucan(Q-) extracted using hot water. The structure of Q+ and Q- were confirmed through Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopies. Q+ contained 41.66 % ß-glucan, 3.93 % protein, 2.12 % uronic acid; Q- contained 37.21 % ß-glucan, 11.49 % protein, and 1.73 % uronic acid. The average molecular weight of Q+(75.37 kDa) was lower than that of Q- (94.47 kDa). Both Q+ and Q- promote RAW264.7 cell proliferation without displaying toxicity. They stimulate RAW264.7 cells through the NF-κB and MAPK signaling pathways, primarily inducing NO and pro-inflammatory cytokines by upregulating CD40 expression. Notably, Q+ exhibited stronger immunostimulatory activity compared to Q-. In summary, the fermentation enrichment method yields higher content of quinoa ß-glucan with increased purity and stronger immunostimulatory properties. Further study of its bioimmunological activity and structure-activity relationship may contribute to the development of new immunostimulants.

Analysis of widely targeted metabolites of quinoa sprouts (Chenopodium quinoa Willd.) under saline-alkali stress provides new insights into nutritional value.

Quinoa sprouts are a green vegetable rich in bioactive chemicals, which have multiple health benefits. However, there is limited information on the overall metabolic profiles of quinoa sprouts and the metabolite changes caused by saline-alkali stress. Here, a UHPLC-MS/MS-based widely targeted metabolomics technique was performed to comprehensively evaluate the metabolic profiles of quinoa sprouts and characterize its metabolic response to saline-alkali stress. A total of 930 metabolites were identified of which 232 showed significant response to saline-alkali stress. The contents of lipids and amino acids were significantly increased, while the contents of flavonoids and phenolic acids were significantly reduced under saline-alkali stress. Moreover, the antioxidant activities of quinoa sprouts were significantly affected by saline-alkali stress. The enrichment analysis of the differentially accumulated metabolites revealed that flavonoid, amino acid and carbohydrate biosynthesis/metabolism pathways responded to saline-alkali stress. This study provided an important theoretical basis for evaluating the nutritional value of quinoa sprouts and the changes in metabolites in response to saline-alkali stress.

Key phytochemicals contributing to the bitterness of quinoa.

Despite its nutritional components and potential health benefits, the bitterness of quinoa seed limits its utilization in the food industry. Saponins are believed to be the main cause of the bitterness, but it is still uncertain which specific compound is responsible. This study aimed to isolate the main components contributing to the bitterness in quinoa seed by solvent extraction and various column chromatography techniques guided by sensory evaluation. Five compounds were identified by mass spectrometry and nuclear magnetic resonance analyses, with the dose-over-threshold factors from 29.03 to 198.89. The results confirmed that triterpenoids are responsible for the bitter taste in quinoa seed, with phytolaccagenic acid derivatives being the primary contributor. Additionally, kaempferol 3-O-(2″, 6″-di-O-α-rhamnopyranosyl)-ß-galactopyranoside (namely mauritianin), was demonstrated for the first time to be associated with the bitterness of quinoa. This study could provide new insight into the bitter compound identification in quinoa.

The beneficial effects of quinoa seed extract supplementation on ram sperm quality following cryopreservation.

Although cryopreservation is a reliable method used in assisted reproduction to preserve genetic materials, it can stimulate the occurrence of oxidative stress, which affects sperm structure and function. This research was conducted to explore the effects of quinoa seed extracts (QSE) on ram sperm quality, oxidative biomarkers, and the gene expression of frozen-thawed ram sperm. Semen samples were diluted in extenders supplemented with 0 (QSE0), 250 (QSE1), 500 (QSE2), 750 (QSE3), and 1000 (QSE4) µg of QSE /mL, and then frozen according to the typical procedure. The findings indicate that the QSE3 and QSE4 groups provided the optimal results in terms of sperm viability and progressive motility. Sperm kinematics were considerably enhanced in the QSE3 group compared to the other groups (P<0.01). QSE (500-1000 µg/mL) significantly decreased the apoptosis-like changes (higher viable and lower apoptotic sperm) in ram sperm (P<0.001). The percentage of live sperm with intact acrosomes was significantly increased, while the percentage of detached and intact acrosomes in live and dead sperm were significantly decreased respectively by the QSE addition (P<0.001). All QSE groups had higher TAC and lower MDA and H2O2 levels than the control group (P<0.001). The expressions of SOD1, CAT, GABPB1, and GPX1 genes in sperm samples were significantly increased, while the CASP3 gene was significantly decreased in all QSE-supplemented samples. Our data suggest that QSE has beneficial effects on sperm quality of cryopreserved ram semen, which are achieved by promoting sperm antioxidant-related genes and reducing apoptosis-related gene.

Technofunctional Properties and Rheological Behavior of Quinoa, Kiwicha, Wheat Flours and Their Mixtures.

Wheat flour is a common raw material in the food industry; however, Andean grains, such as quinoa and kiwicha, are gaining popularity due to their quality proteins, fiber, and bioactive compounds. A trend has been observed toward the enrichment of products with these Andean flours, with them even being used to develop gluten-free foods. However, evaluating interactions between raw materials during industrial processes can be complicated due to the diversity of inputs. This study focused on evaluating the technofunctional and rheological properties of wheat, quinoa and kiwicha flours using a simple lattice mixture design. Seven treatments were obtained, including pure flours and ternary mixtures. Analyses of particle size distribution, water absorption index, subjective water absorption capacity, soluble material index, swelling power, apparent density and physicochemical properties were performed. Additionally, color analysis, photomicrographs and Raman spectroscopy were carried out. The results indicate significant differences in properties such as particle size, water absorption and rheological properties between the flours and their mixtures. Variations in color and microstructure were observed, while Raman spectroscopy provided information on molecular composition. These findings contribute to the understanding of the behavior of Andean flours in baking and pastry making, facilitating their application in innovative food products.

Biotechnological, Nutritional, and Therapeutic Applications of Quinoa (Chenopodium quinoa Willd.) and Its By-Products: A Review of the Past Five-Year Findings.

This study aimed to provide an updated critical review of the nutritional, therapeutic, biotechnological, and environmental aspects involved in the exploitation of Chenopodium quinoa Willd and its biowastes. Special attention was devoted to investigations of the therapeutic and nutritional properties of different parts and varieties of quinoa as well as of the use of the biowaste resulting from the processing of grain. Studies published from 2018 onward were prioritized. Extracts and fractions obtained from several Chenopodium quinoa matrices showed antioxidant, antidiabetic, immunoregulatory, neuroprotective, and antimicrobial effects in in vitro and in vivo models and some clinical studies. The activities were attributed to the presence of phytochemicals such as polyphenols, saponins, peptides, polysaccharides, and dietary fibers. Quinoa wastes are abundant and low-cost sources of bioactive molecules for the development of new drugs, natural antioxidants, preservatives, dyes, emulsifiers, and carriers for food and cosmetics applications. Among the demands to be fulfilled in the coming years are the following: (1) isolation of new bioactive phytochemicals from quinoa varieties that are still underexploited; (2) optimization of green approaches to the sustainable recovery of compounds of industrial interest from quinoa by-products; and (3) well-conducted clinical trials to attest safety and efficacy of extracts and compounds.

Protein extracts from amaranth and quinoa as novel fining agents for red wines.

Due to allergenic concerns, only pea, potato, and wheat proteins have been approved as alternatives for replacing animal-based fining agents in wines. In pursuit of other substitutes, this work aimed to determine the fining ability of amaranth (Amaranthus caudatus L.) proteins (AP) in red wine, compared to quinoa (Chenopodium quinoa Willd.) (QP) and a commercial pea protein. Phenolic and volatile composition, as well as color characteristics, were analyzed. AP was as effective as QP at decreasing condensed tannins, with AP at 50 g/hL being the most effective treatment (25.6% reduction). QP and AP produced a minor or no statistical change in the total anthocyanins and wine color intensity. They reduced the total ester concentration, but the total alcohols remained unchanged. The outcomes of AP and QP were similar, and sometimes better than the pea proteins, thus suggesting that they could be promising options for the development of novel fining agents.

Change of physiochemical characteristics, nutritional quality, and volatile compounds of Chenopodium quinoa Willd. during germination.

The impacts of varying germination periods (0-72 h) on morphological properties, proximate composition, amino acid profile, GABA levels, antioxidant attributes, polyphenol content (both free and bound), and volatile compounds of quinoa were evaluated. Germination significantly increased the content of fiber, amino acids, GABA, polyphenols, and in-vitro antioxidant activities in quinoa. The optimal nutritional quality and antioxidant capacity of quinoa were observed during the 36-72 h germination period. We examined the dynamics of 47 phenolic compounds in quinoa during germination and noted a substantial rise in free phenolic acids and bound flavonoids post-germination. A total of 53 and 84 volatile compounds were respectively identified in ungerminated quinoa and germinated quinoa. It was found that the germination period of 24-48 h contributed to reducing the presence of undesirable flavors. TEM analysis revealed significant structural damage to the ultrastructure and relaxation of the cell wall in germinated quinoa grains.

Refining quinoa storage stability through microwave-induced structural alterations and activity suppression of key enzymes.

This study investigated the effects of microwave on preserving the quality of quinoa during storage. Quinoa treated with 9W/60s exhibited a significant decrease in fatty acid values compared to hot air treatment. Microwave effectively delayed lipid oxidation during quinoa storage by suppressing the increase in peroxide values. MDA gradually accumulated from peroxides during storage, reaching its peak at 0.423 µmol/L in the second week. Microwave disrupted the original hydrogen bonds in lipase, causing the unwinding of the α-helix and resulting in the loss of its regular structure. Microwave reduced the stability of the ß-sheet structure in lipoxygenase, breaking the natural secondary structure composition. The observed fluorescence and UV spectra features were similar, indicating that microwave alter the peptide chain of the enzyme's skeletal structure, increasing the exposure of hydrophobic chromophores. These results indicated the potential of microwave to enhance the stability of quinoa during storage.

Physicochemical properties of esterified/crosslinked quinoa starches and their influence on bread quality.

BACKGROUND: Starch is the main component of quinoa seeds. However, quinoa starch has poor solubility in cold water and poor mechanical resistance and is easily aged, which limit its application. Therefore, modification of its structure to improve its functional properties is necessary. RESULTS: This research used acetic anhydride and sodium trimetaphosphate to modify the structure of starch molecules and investigated their influence on bread quality. The results showed that both esterification and crosslinking prevented the aggregation behavior of starch molecules. Moreover, they both decreased the gelatinization enthalpy change and relative crystallinity of the starch. Compared with native starch, modification significantly decreased the gelatinization temperature from 57.01 to 52.01 °C and the esterified starch exhibited the lowest enthalpy change with a 44.2% decrease. Modified starch increased the specific volume and decreased the hardness and chewiness of bread. Modification did not influence the moisture content in bread but impacted the water retention capacity, depending on the degree of modification. Low and medium degrees of modification improved the water retention capacity during storage. By contrast, a high degree of modification (10 g kg-1 crosslinking agent) decreased the water retention capacity. The dually modified quinoa starch (esterified and crosslinked) showed no influence on the textural properties of bread. CONCLUSION: This study demonstrated that both esterification and crosslinking significantly improved the functional properties of quinoa starch. Crosslinked or esterified quinoa starches have the potential to improve the textural properties of bakery products. © 2024 Society of Chemical Industry.

Nutritional composition of quinoa leafy greens: An underutilized plant-based food with the potential of contributing to current dietary trends.

Quinoa (Chenopodium quinoa Willd.) leafy greens (QLGs) are plant-based foods of high nutritional value that have been scarcely studied. In this work, the nutritional and functional composition of three QLGs varieties was evaluated. A protein content higher than 35 g 100 g-1 dw with a well-balanced essential amino acid composition was found making them a good source of vegetable protein. In addition, elevated contents of dietary fibre and minerals, higher than those detected in quinoa seeds and other leafy vegetables, were found. The lipid profile showed higher contents of linoleic (C18:2, ω6) (20.2 %) and linolenic acids (C18:3, ω3) (52.8 %) with low ω6/ ω3 ratios (∼0.4/1). A total sugar content <1 g 100 g-1 dw was found for all varieties tested, lower than that obtained in seeds. The saponin content varied between 0.76 and 0.87 %. Also, high values of total phenolic compounds (969.8-1195.4 mg gallic acid 100 g-1), mainly hydroxycinnamic acids and flavonoids, and great antioxidant activities (7.64-8.90 g Trolox kg-1) were found. Multivariate analysis here used allowed us to classify the samples according to the quinoa variety evaluated, and the sequential stepwise multiple regression applied revealed that the PUFA and sucrose contents negatively influenced the protein content while the palmitic acid content affected positively this parameter. Overall, this study shows that QLGs are promising nutritious and functional plant-based foods supporting the necessity of promoting their cultivation, commercialization, and consumption.

Transcriptome Analysis Reveals the Mechanisms of Accumulation and Conversion of Folate Derivatives during Germination of Quinoa (Chenopodium quinoa Willd.) Seeds.

Folate was enriched during quinoa germination, while molecular mechanisms were not well understood. In this study, three quinoa varieties were selected for germination, and changes in substrate content and enzyme activity of the folate biosynthesis pathway were monitored. 5-Methyltetrahydrofolate (5-CH3-THF) and 5-formyltetrahydrofolate (5-CHO-THF) were significantly enriched in quinoa sprouts. Among the selected varieties, QL-2 exhibited the lowest content of the oxidation product MeFox and the highest total folate content. Based on transcriptome analysis, the p-ABA branch was found to be crucial for folate accumulation, while the pterin branch served as a key control point for the one carbon pool by folate pathway, which limited further folate biosynthesis. In the one carbon pool by folate pathway, genes CqMTHFR and CqAMT significantly contributed to the enrichment of 5-CH3-THF and 5-CHO-THF. Findings gained here would facilitate the potential application of quinoa sprouts as an alternative strategy for folate supplementation.

Flours from fermented lentil and quinoa grains as ingredients with new techno-functional properties.

The need to provide novel, nutritious plant-based products requires seeking high-value, sustainable protein sources, like quinoa and lentils, having an increased digestibility and lacking antinutrients. Fungal fermentation has evidenced enhanced nutritional value of flours obtained from these grains. However, research into techno-functional properties, essential to the new product development, is lacking. This study investigated the techno-functional properties of flours made from lentil and quinoa after fermenting them with Pleurotus ostreatus and subjecting them to two drying techniques (lyophilisation and hot air drying). In both cases, the fermentation led to noteworthy improvements in swelling and water holding capacity, especially in those lyophilised than those dried. In contrast, the emulsifying, foaming, thickening, and gelling capacities decreased significantly. The loss of abilities was more severe for dried grains than for lyophilized ones. The thermomechanical analysis of the fermented flours showed lower thickening and gelling potential compared to untreated flours. Microscopy images revealed that the state and structure of starch granules were affected by both fermentation and drying processes. Starch granules in lentils were partly pre-gelatinised and trapped in the cotyledon cell, resulting in limited thickening and gelling abilities. In contrast, in quinoa, starch underwent pre-gelatinisation and retrogradation during the fermentation process, promoting the production of resistant starch and increasing fibre content. This study presents the potential of treated flours as ingredients possessing unique attributes compared to protein and fibre-rich conventional products.

Structural, physicochemical properties and noodle-making potential of quinoa starch and type 3, type 4, and type 5 quinoa resistant starch.

This study prepared type 3, type 4, and type 5 quinoa resistant starch (QRS3, QRS4, and QRS5) from quinoa starch (QS), compared their structural and physicochemical properties and evaluated their noodle-making potential. The results showed that the molecular weight of QRS3 decreased, the number of short-chain molecules increased, and its crystal type changed to B-type after gelatinization, enzymatic hydrolysis, and retrogradation. QRS4 is a phosphorylated cross-linked starch, with a surface morphology, particle size range, and crystal type similar to QS, but displaying modified thermodynamic properties. QRS5 is a complex of amylose and palmitic acid. It displays typical V-type crystals, mainly composed of long chain molecules and primarily exhibits a block morphology. The noodles prepared by replacing 20 % wheat flour with QS, QRS3 and QRS5 have higher hardness and are suitable for people who like elasticity and chewiness. QRS4 noodles are softer and suitable for people like elderly and infants who prefer soft foods. In conclusion, significant differences were evident between the fine structures, crystal types, physicochemical properties and potential applications of QS and the three QRSs. The results may expand the application of QS and QRS in the food and pharmaceutical industries.

The potential of young vegetative quinoa (Chenopodium quinoa) as a new sustainable protein-rich winter leafy crop under Mediterranean climate.

The demand for protein products has significantly risen in the last few years. In western countries, animals are the primary source of protein; however, plants could take a share of this market due to lower production costs, among other advantages such as a lower environmental footprint. Quinoa (Chenopodium quinoa Willd.) is a well-known but under-utilized protein-rich crop, commonly cultivated for grain production. These plants were recently evaluated for their use as a non-traditional, green leafy crop. Here we assessed the potential of young vegetative quinoa as a new sustainable winter leafy crop in Israel-serving as a model for Mediterranean semi-arid regions, by evaluating yield, protein content and quality. Five quinoa accessions were sown on three winter sowing dates over two consecutive years. Plants were harvested when they reached 10% dry matter (DM). DM yield ranged between 574 and 1,982 kg ha-1 and was generally higher in the second year. Protein content ranged from 14.4-34% and was generally higher in the first year. Protein yield ranged from 111-471 kg ha-1 and was greatest on the December sowing date. DM and protein yields were positively correlated with plant density. Protein content was negatively correlated with plant density and DM yield. Our findings show that 200 g DM of young vegetative quinoa can meet the protein and most essential amino acid requirements for a 70 kg human adult. Prospects for cultivating young vegetative quinoa in Mediterranean countries as a new sustainable, protein-rich winter leafy crop are therefore high, as supported by its high protein yields and quality, and its requirement for only scant irrigation. Further studies should examine economic and other agrotechnical parameters toward the geographical distribution and expansion of young vegetative quinoa cultivation.

Effect of Starch on the Solubility of Quinoa Protein Isolates during Heat Treatment.

There is increasing interest in developing quinoa products due to their unique nutritional value. Starch and protein are the primary components in quinoa, and the interaction between them affects the quality of quinoa products. This study extracted the starch and protein from quinoa and simulated the thermal processing of quinoa to investigate the effects of starch on the solubility and structure of quinoa protein isolates during heat treatment. The structure of quinoa protein isolates was characterized by fluorescence spectroscopy, Fourier transform infrared spectroscopy, laser particle size analysis, and scanning electron microscopy. The results showed that starch decreased protein solubility, and the maximum solubility was obtained after heating for 5 min. After starch addition during heat treatment, the surface charge distribution of protein changed, the degree of protein aggregation increased, the particle size of proteins increased, the thermal stability increased, and the ß-sheet ratio of the proteins increased, suggesting that the protein structure is more ordered, which is the structural foundation of protein solubility decreasing. The research about the interaction between starch and protein and the effects on the solubility of protein could provide a reference for quinoa products processing.

Influence of in vitro gastrointestinal digestion and colonic fermentation on carbonyl scavenging capacity of fiber-bound polyphenols from quinoa.

Whole grain insoluble dietary fiber (IDF) is a good source of bound-form polyphenols. In the present study, insoluble dietary fiber rich in bound polyphenols (BP-IDF) from quinoa, rye and wheat was prepared. The carbonyl scavenging capacities of these three BP-IDFs and the effects of in vitro gastrointestinal (GI) digestion and colonic fermentation on their scavenging activities were studied. The results indicated that the fiber-bound polyphenols from quinoa showed the highest carbonyl scavenging capacity compared to those from rye and wheat. After colonic fermentation, more than 73% of the bound polyphenols were still retained in the fermented residues of the quinoa BP-IDF. The fiber-bound polyphenols in the GI-digested residues of quinoa retained considerable carbonyl scavenging activities. During the fermentation process, the residual fiber-bound polyphenols in the fermented residues still scavenged 35.8% to 45.2% of methylglyoxal, 19.3% to 25.4% of glyoxal, 50.7% to 60.5% of acrolein and 5.2% to 9.7% of malondialdehyde, showing a critical role in the scavenging of carbonyl compounds compared to the released and metabolized polyphenols. These findings confirm the capacity of fiber-bound polyphenols from three whole grains to scavenge carbonyls during in vitro digestion and fermentation processes, suggesting that they could be used as functional ingredients to maintain continuous defenses against carbonyls along the digestive tract.