Do red and yellow autumn leaves make use of different photoprotective strategies during autumn senescence?

Our goal was to determine whether anthocyanin-producing species (red) use different photoprotective strategies to cope with excess light during fall senescence compared with non-anthocyanin-producing species (yellow). In a previous study, we found that a yellow species retained the photoprotective PsbS protein in late autumn, while a red species did not. Specifically, we tested the hypothesis that red species make less use of zeaxanthin and PsbS-mediated thermal dissipation, as they rely on anthocyanins for photoprotection. We monitored four red (Acer ginnala, Rhus typhnia, Parenthocissus quinquefolia, Viburnum dentatum) and four yellow species (Acer negundo, Ostrya virginiana, Vitis riparia, Zanthoxylum americanum) throughout autumn senescence and analyzed pigments, protein content, and chlorophyll fluorescence. We found yellow species retained the PsbS protein at higher levels, and had higher dark retention of zeaxanthin in late autumn relative to red species. All species retained lutein and the pool of xanthophyll cycle pigments in higher amounts than other carotenoids in late autumn. Our data support the hypothesis that red species use anthocyanins as a photoprotective strategy during autumn senescence, and therefore make less use of PsbS and zeaxanthin-mediated thermal dissipation. We also found species-specific variation in the particular combination of photoprotective strategies used.

Zeaxanthin dipalmitate-enriched wolfberry extract improves vision in a mouse model of photoreceptor degeneration.

Zeaxanthin dipalmitate (ZD) is a chemical extracted from wolfberry that protects degenerated photoreceptors in mouse retina. However, the pure ZD is expensive and hard to produce. In this study, we developed a method to enrich ZD from wolfberry on a production line and examined whether it may also protect the degenerated mouse retina. The ZD-enriched wolfberry extract (ZDE) was extracted from wolfberry by organic solvent method, and the concentration of ZD was identified by HPLC. The adult C57BL/6 mice were treated with ZDE or solvent by daily gavage for 2 weeks, at the end of the first week the animals were intraperitoneally injected with N-methyl-N-nitrosourea to induce photoreceptor degeneration. Then optomotor, electroretinogram, and immunostaining were used to test the visual behavior, retinal light responses, and structure. The final ZDE product contained ~30mg/g ZD, which was over 9 times higher than that from the dry fruit of wolfberry. Feeding degenerated mice with ZDE significantly improved the survival of photoreceptors, enhanced the retinal light responses and the visual acuity. Therefore, our ZDE product successfully alleviated retinal morphological and functional degeneration in mouse retina, which may provide a basis for further animal studies for possible applying ZDE as a supplement to treat degenerated photoreceptor in the clinic.

Microbial chassis as the platform for production of dihydroxy xanthophyll-based carotenoids: an overview of recent advances in biomanufacturing.

Physiological and environmental cues prompt microbes to synthesize diverse carotenoids, including dihydroxy xanthophylls, facilitating their adaptation and survival. Lutein and its isomeric counterpart, zeaxanthin, are notable dihydroxy xanthophylls with bioactive properties such as antioxidative, anti-inflammatory, anticancer, and neuroprotective effects, particularly beneficial for human ocular health. However, global natural resources for co-producing lutein and zeaxanthin are scarce, with zeaxanthin lacking commercial sources, unlike lutein sourced from marigold plants and microalgae. Traditionally, dihydroxy xanthophyll production primarily relies on petrochemical synthetic routes, with limited biological sourcing reported. Nonetheless, microbiological synthesis presents promising avenues as a commercial source, albeit challenged by low dihydroxy xanthophyll yield at high cell density. Strategies involving optimization of physical and chemical parameters are essential to achieve high-quality dihydroxy xanthophyll products. This overview briefly discusses dihydroxy xanthophyll biosynthesis and highlights recent advancements, discoveries, and industrial benefits of lutein and zeaxanthin production from microorganisms as alternative biofactories.

Lutimonas zeaxanthinifaciens sp. nov., a zeaxanthin-producing marine bacterium isolated from coastal sediment.

A Gram-stain-negative, yellow-pigmented, strictly aerobic, non-flagellated, motile by gliding, rod-shaped bacterium, designated strain YSD2104T, was isolated from a coastal sediment sample collected from the southeastern part of the Yellow Sea. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain YSD2104T was closely related to three type strains, Lutimonas vermicola IMCC1616T (97.4 %), Lutimonas saemankumensis SMK-142T (96.9 %), and Lutimonas halocynthiae RSS3-C1T (96.8 %). Strain YSD2104T has a single circular chromosome of 3.54 Mbp with a DNA G+C content of 38.3 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain YSD2104T and the three type strains (L. vermicola IMCC1616 T, L. saemankumensis SMK-142T, and L. halocynthiae RSS3-C1T) were 74.0, 86.2 and 73.6 %, and 17.9, 30.3 and 17.8 %, respectively. Growth was observed at 20-30 °C (optimum, 30 °C), at pH 6.5-8.5 (optimum, pH 7.0), and with NaCl concentrations of 1.5-3.5 % (optimum, 2.5 %). The major carotenoid was zeaxanthin, and flexirubin-type pigment was not produced. The major respiratory quinone was menaquinone-6. The major fatty acids (>10 %) were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH, summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), and summed feature 9 (iso-C17 : 1 ω9c and/or 10-methyl C16 : 0). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, two unidentified aminolipids, and eight unidentified lipids. Conclusively, based on this polyphasic approach, we classified strain YSD2104T (=KCTC 102008T=JCM 36287T) as representing a novel species of the genus Lutimonas and proposed the name Lutimonas zeaxanthinifaciens sp. nov.

Bioavailability of Lutein from Marigold Flowers (Free vs. Ester Forms): A Randomised Cross-Over Study to Assess Serum Response and Visual Contrast Threshold in Adults.

Lutein (Lut) and zeaxanthin (Zeax) are found in the blood and are deposited in the retina (macular pigment). Both are found in the diet in free form and esterified with fatty acids. A high intake and/or status is associated with a lower risk of chronic diseases, especially eye diseases. There is a large global demand for Lut in the dietary supplement market, with marigold flowers being the main source, mainly as lutein esters. As the bioavailability of Lut from free or ester forms is controversial, our aim was to assess the bioavailability of Lut (free vs. ester) and visual contrast threshold (CT). Twenty-four healthy subjects (twelve women, twelve men), aged 20-35 and 50-65 years, were enrolled in a cross-sectional study to consume 6 mg lutein/day from marigold extract (free vs. ester) for two months. Blood samples were taken at baseline and after 15, 40, and 60 days in each period. Serum Lut and Zeax were analysed using HPLC, and dietary intake was determined with a 7-day food record at the beginning of each period. CT, with and without glare, was at 0 and 60 days at three levels of visual angle. Lut + Zeax intake at baseline was 1.9 mg/day, and serum lutein was 0.36 µmol/L. Serum lutein increased 2.4-fold on day 15 (up to 0.81 and 0.90 µmol/L with free and ester lutein, respectively) and was maintained until the end of the study. Serum Zeax increased 1.7-fold. There were no differences in serum Lut responses to free or ester lutein at any time point. CT responses to lutein supplementation (free vs. ester) were not different at any time point. CT correlated with Lut under glare conditions, and better correlations were obtained at low frequencies in the whole group due to the older group. The highest correlations occurred between CT at high frequency and with glare with serum Lut and Lut + Zeax. Only in the older group were inverse correlations found at baseline at a high frequency with L + Z and with Lut/cholesterol and at a low frequency with Lut/cholesterol. In conclusion, daily supplementation with Lut for 15 days significantly increases serum Lut in normolipemic adults to levels associated with a reduced risk of age-related eye disease regardless of the chemical form of lutein supplied. Longer supplementation, up to two months, does not significantly alter the concentration achieved but may contribute to an increase in macular pigment (a long-term marker of lutein status) and thus improve the effect on visual outcomes.

Association between carotenoid intake and periodontitis in diabetic patients.

This study aimed to evaluate the association between dietary carotenoid intake and periodontitis in diabetic patients. Data on diabetic patients were collected from the National Health and Nutrition Examination Survey (NHANES) 2009-2014 for this cross-sectional study. Dietary intake of carotenoids was assessed through the first 24-hour dietary recall interview. Full-mouth periodontal examinations were conducted by trained dental examiners. Subgroup analysis was conducted in terms of age, gender, the number of missing teeth, cardiovascular disease, smoking, and anti-diabetic drugs. Totally 1914 diabetic patients were included, with 1281 (66.93%) in the periodontitis group. After adjusting for age, gender, race, education, smoking, dental implants, hepatitis, and the number of missing teeth, α-carotene intake ≥55.82 mcg was associated with lower odds of periodontitis than α-carotene intake <55.82 mcg [OR = 0.70, 95% CI: 0.53-0.91, P = 0.010]; lutein and zeaxanthin intake ≥795.95 mcg was associated with decreased odds of periodontitis than lutein and zeaxanthin intake <795.95 mcg (OR = 0.75, 95%CI: 0.57-0.98, P = 0.039). The association between carotenoid intake and periodontitis varied across different subpopulations. In diabetes, dietary intake of α-carotene and lutein and zeaxanthin was inversely associated with the odds of periodontitis, which may facilitate clinical periodontitis management.

Systems Metabolic Engineering for Efficient Violaxanthin Production in Yeast.

Violaxanthin is a plant-derived orange xanthophyll with remarkable antioxidant activity that has wide applications in various industries, such as food, agriculture, and cosmetics. In addition, it is the key precursor of important substances such as abscisic acid and fucoxanthin. Saccharomyces cerevisiae, as a GRAS (generally regarded as safe) chassis, provides a good platform for producing violaxanthin production with a yield of 7.3 mg/g DCW, which is far away from commercialization. Herein, an integrated strategy involving zeaxanthin epoxidase (ZEP) source screening, cytosol redox state engineering, and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration was implemented to enhance violaxanthin production in S. cerevisiae. 58aa-truncated ZEP from Vitis vinifera exhibited optimal efficiency in an efficient zeaxanthin-producing strain. The titer of violaxanthin gradually increased by 17.9-fold (up to 119.2 mg/L, 15.19 mg/g DCW) via cytosol redox state engineering and NADPH supplementation. Furthermore, balancing redox homeostasis considerably improved the zeaxanthin concentration by 139.3% (up to 143.9 mg/L, 22.06 mg/g DCW). Thus, the highest reported titers of violaxanthin and zeaxanthin in S. cerevisiae were eventually achieved. This study not only builds an efficient platform for violaxanthin biosynthesis but also serves as a useful reference for the microbial production of xanthophylls.

Effect of Antioxidant Supplementation on Macular Pigment Optical Density and Visual Functions: A Systematic Review and Network Meta-Analysis of Randomized Controlled Trials.

Antioxidants are bioactive molecules that function to scavenge free radicals and balance oxidative stress. Although all antioxidants can act as reactive oxygen species scavengers, their efficacy on eye health may vary. Moreover, the comparative effectiveness and potential additive effect between groups of antioxidants, hitherto, have not been systematically studied. A systematic review and network meta-analysis were conducted to investigate the comparative or additive effect of dietary antioxidant supplements on eye health. Four databases (PubMed, Embase, CINAHL, and Cochrane) were searched, and relevant randomized controlled trials were identified. Out of 60 articles selected for systematic review, 38 were included in the network meta-analysis, categorized into 8 distinct antioxidant-supplemented groups and placebo. All groups significantly increased macular pigment optical density and contrast sensitivity at low spatial frequency, whereas only the antioxidant mixture + lutein (L) + fatty acid combination exhibited significant improvements in visual acuity (hazard ratio = -0.15; 95% confidence interval: -0.28, -0.02) and L + zeaxanthin combination for photostress recovery time (hazard ratio = -5.75; 95% confidence interval: -8.80, -1.70). Especially, the L + zeaxanthin + fatty acid combination was ranked best for macular pigment optical density (surface under the cumulative ranking: 99.3%) and second best for contrast sensitivity at low spatial frequency (67.7%). However, these findings should be interpreted with caution due to low quality of evidence, primarily influenced by indirectness and potential publication bias. Overall, antioxidant supplementation was estimated to improve eye health parameters, whereas different combinations of antioxidants may also have varying effects on improving visual health from multiple perspectives. This study was registered at PROSPERO as CRD42022369250.

Continuous microalgal culture module and method of culturing microalgae containing macular pigment.

This study established and investigated continuous macular pigment (MP) production with a lutein (L):zeaxanthin (Z) ratio of 4-5:1 by an MP-rich Chlorella sp. CN6 mutant strain in a continuous microalgal culture module. Chlorella sp. CN6 was cultured in a four-stage module for 10 days. The microalgal culture volume increased to 200 L in the first stage (6 days). Biomass productivity increased to 0.931 g/L/day with continuous indoor white light irradiation during the second stage (3 days). MP content effectively increased to 8.29 mg/g upon continuous, indoor white light and blue light-emitting diode irradiation in the third stage (1 day), and the microalgal biomass and MP concentrations were 8.88 g/L and 73.6 mg/L in the fourth stage, respectively. Using a two-step MP extraction process, 80 % of the MP was recovered with a high purity of 93 %, and its L:Z ratio was 4-5:1.

Zeaxanthin impairs angiogenesis and tumor growth of glioblastoma: An in vitro and in vivo study.

OBJECTIVES: To investigate the therapeutic effects of Zeaxanthin (Zea), one of the oxidized xanthophyll carotenoids belonging to the isoprenoids, on inhibiting the angiogenesis and tumor growth of glioblastoma (GBM) via an in vitro and in vivo study. METHODS: The effects of Zea on the proliferation, adhesion, migration and invasion of human GBM cell lines were detected by cell proliferation assay, cell adhesion assay and Transwell assay. The effect of Zea on angiogenesis was detected by rat aortic ring assay and human umbilical vein endothelial cells (HUVEC) in vitro tube formation assay. The effects of Zea on PARP, Caspase 3 and VEGFR2 phosphorylation as well as VEGFR2's downstream signaling pathway were detected by Western blot. The in vivo human GBM xenograft mouse model was employed to study the therapeutic efficacy of Zea. RESULTS: Zea impaired the proliferation, adhesion, migration and invasion of U87 and U251 cells as well as HUVECs. Rat aortic ring experiments displayed Zea significantly inhibited angiogenesis during VEGF-induced microvascular germination. In vitro and in vivo vascular experiments verified that Zea inhibited VEGF-induced HUVEC proliferation and capillary-like tube formation. Additionally, Zea induced GBM cells apoptosis via increasing the expression of cleaved PARP and Caspase 3. In HUVECs and U251 GBM cells, Zea down-regulated VEGF-induced activation of the VEGFR2 kinase pathway. Meanwhile the expression of p-AKT, p-ERK, p-STAT3 and FAK were all attenuated in U251 cells. Moreover, the effects of Zea on GBM cells proliferation could be blocked by VEGFR2 kinase inhibitor SU5408. These results suggest that Zea may hinder GBM angiogenesis and tumor growth through down-regulating a cascade of oncogenic signaling pathways, both through the inhibition of angiogenesis and the anti-tumor mechanism of a direct cytotoxic effect. Besides, Zea inhibits GBM angiogenesis and tumor growth exemplified through a xenograft mouse model in vivo. CONCLUSION: Zea impairs angiogenesis and tumor growth of GBM both in vitro and in vivo. It can be declared that Zea is a potential valuable anticancer candidate for the future treatment strategy of GBM.

Association between carotenoids and the prevalence of chronic obstructive pulmonary disease in the United States.

BACKGROUND: Previous studies mainly concentrated on examining the correlation between single carotenoids and Chronic obstructive pulmonary disease (COPD). However, these findings have been inconsistent. OBJECTIVES: This study aimed to evaluate both the individual and overall associations of carotenoids with the prevalence of COPD. METHODS: This study comprised 2,939 participants chosen from the National Health and Nutrition Examination Survey (NHANES) 2017-2018. The logistic regression, quantile-based G-computation regression (qgcomp), and Bayesian kernel machine regression (BKMR) models were employed to explore the association between carotenoids and the prevalence of COPD. Mediation analyses were also conducted to explore the underlying mechanism of carotenoids on COPD. RESULTS: Individuals diagnosed with COPD had significantly lower serum carotenoid concentrations than those without COPD. We found a negative relationship between combined carotenoids and the prevalence of COPD, and lutein and zeaxanthin and alpha cryptoxanthin were identified as the main contributors to this negative association. Moreover, eosinophil acted as a mediator in the relationship between lutein and zeaxanthin, alpha cryptoxanthin, and the prevalence of COPD, with mediating proportions of 2.75 % and 3.67 %. CONCLUSION: A negative association was observed between combined carotenoids and COPD prevalence, with lutein and zeaxanthin, and alpha cryptoxanthin identified as the main contributors. Eosinophils could potentially mediate the association between carotenoids and COPD.

Combined high light and salt stress enhances accumulation of PsbS and zeaxanthin in Chlamydomonas reinhardtii.

The performance and acclimation strategies of Chlamydomonas reinhardtii under stress conditions are typically studied in response to single stress factors. Under natural conditions, however, organisms rarely face only one stressor at a time. Here, we investigated the impact of combined salt and high light stress on the photoprotective response of C. reinhardtii. Compared to the single stress factors, the combination of both stressors decreased the photosynthetic performance, while the activation of energy dissipation remained unaffected. However, the PsbS protein was strongly accumulated and the conversion of violaxanthin to zeaxanthin was enhanced. These results support an important photoprotective function of PsbS and zeaxanthin independently of energy dissipation under combined salt and high light stress in C. reinhardtii.

Valorization of Nannochloropsis oceanica for integrated co-production of violaxanthin cycle carotenoids.

The development of integrated co-production of multiple high-purity carotenoids from microalgal cells holds considerable significance for the valorization of microalgae. In this study, the economical microalga Nannochloropsis oceanica was identified as an accumulator of violaxanthin cycle carotenoids, including violaxanthin, antheraxanthin, and zeaxanthin. Notably, a novel and competent approach for the integrated co-production of violaxanthin cycle carotenoids was explored, encompassing four steps: microalgal cultivation, solvent extraction, octadecylsilyl open-column chromatography, and ethanol precipitation. Under optimal co-production conditions, the purities of the obtained violaxanthin, antheraxanthin, and zeaxanthin all exceeded 92%, with total recovery rates of approximately 51%, 40%, and 60%, respectively. Utilizing nuclear magnetic resonance techniques, the purified violaxanthin, antheraxanthin, and zeaxanthin were identified as all-trans-violaxanthin, all-trans-antheraxanthin, and all-trans-zeaxanthin, respectively. This method held significance for the multiproduct biorefinery of the microalga N. oceanica and carried potential future implications for the violaxanthin cycle carotenoids.

Deep learning-based correction of cataract-induced influence on macular pigment optical density measurement by autofluorescence spectroscopy.

PURPOSE: Measurements of macular pigment optical density (MPOD) using the autofluorescence spectroscopy yield underestimations of actual values in eyes with cataracts. Previously, we proposed a correction method for this error using deep learning (DL); however, the correction performance was validated through internal cross-validation. This cross-sectional study aimed to validate this approach using an external validation dataset. METHODS: MPODs at 0.25°, 0.5°, 1°, and 2° eccentricities and macular pigment optical volume (MPOV) within 9° eccentricity were measured using SPECTRALIS (Heidelberg Engineering, Heidelberg, Germany) in 197 (training dataset inherited from our previous study) and 157 eyes (validating dataset) before and after cataract surgery. A DL model was trained to predict the corrected value from the pre-operative value using the training dataset, and we measured the discrepancy between the corrected value and the actual postoperative value. Subsequently, the prediction performance was validated using a validation dataset. RESULTS: Using the validation dataset, the mean absolute values of errors for MPOD and MPOV corrected using DL ranged from 8.2 to 12.4%, which were lower than values with no correction (P < 0.001, linear mixed model with Tukey's test). The error depended on the autofluorescence image quality used to calculate MPOD. The mean errors in high and moderate quality images ranged from 6.0 to 11.4%, which were lower than those of poor quality images. CONCLUSION: The usefulness of the DL correction method was validated. Deep learning reduced the error for a relatively good autofluorescence image quality. Poor-quality images were not corrected.

Association between dietary carotenoid intakes and abdominal aortic calcification in adults: National Health and Nutrition Examination Survey 2013-2014.

OBJECTIVE: Abdominal aortic calcification (AAC) is an important marker of subclinical atherosclerosis and a predictor of cardiovascular disease. This study aims to explore the association between carotenoid intakes and AAC. METHODS: We included 2889 participants from the National Health and Nutrition Examination Survey (NHANES). Dietary carotenoid intakes were obtained through 24-h dietary recall interviews. Severe AAC was defined as a Kauppila score > 5. The main analysis utilizes logistic and restricted cubic spline models. RESULT: Severe AAC was detected in 378 (13.08%) participants. In fully adjusted models, the odds ratios (OR) with 95% confidence intervals (CI) of α-carotene, ß-carotene, ß-cryptoxanthin, lycopene, lutein with zeaxanthin and total carotenoid intakes for individuals with severe AAC were 0.53 (0.23-0.77), 0.39 (0.19-0.80), 0.18 (0.05-0.62), 0.40 (0.20-0.78), 0.53 (0.32-0.88) and 0.38 (0.18-0.77) in the highest versus lowest quartile intake, respectively. Dose-response analyses revealed that all of the carotenoids were associated with decreased risk of severe AAC in a nonlinear trend. Total carotenoid intakes of at least 100ug/kg/day were associated with decreased odds for severe AAC. CONCLUSION: α-carotene, ß-carotene, ß-cryptoxanthin, lycopene, lutein with zeaxanthin and total carotenoids were inversely associated with the risk of severe AAC in adults.

Lutein and Zeaxanthin Supplementation Improves Dynamic Visual and Cognitive Performance in Children: A Randomized, Double-Blind, Parallel, Placebo-Controlled Study.

INTRODUCTION: Supplementation with dietary neuro-pigments lutein (L) and zeaxanthin (Z) has been shown to improve many aspects of visual and cognitive function in adults. In this study, we tested whether a similar intervention could improve such outcomes in preadolescent children. METHODS: Sixty children (age range 5-12 years) were randomized in a 2:1 ratio in this double-blind, placebo-controlled clinical trial. Subjects were supplemented with gummies containing either a combination of 10 mg lutein and 2 mg zeaxanthin (LZ) or placebo for 180 days. Macular pigment optical density (MPOD) was the primary endpoint. The secondary endpoints included serum levels of L and Z, and brain-derived neurotrophic factor (BDNF), critical flicker fusion (CFF), eye strain and fatigue using visual analogue scales (VAS), Children's Sleep Habits Questionnaire-Abbreviated (CSHQ-A), and Creyos Health cognitive domains like attention, focus/concentration, episodic memory and learning, visuospatial working memory, and visuospatial processing speed. Safety was assessed throughout the study on the basis of physical examination, vital signs, clinical laboratory tests, and monitoring of adverse events. RESULTS: The LZ group showed significant increases in MPOD at all visits post-supplementation, with significant increases as early as day 42 compared to placebo. The LZ group showed significant increases in serum lutein levels, reduced eye strain and fatigue, and improved cognitive performance (focus, episodic memory and learning, visuospatial working memory) at days 90 and 180 compared to placebo. Further, the LZ group showed significant increases in processing speed (CFF), attention, visuospatial processing, and serum Z and BDNF levels on day 180 compared to placebo. No safety concerns were observed. CONCLUSIONS: Supplementing LZ resulted in increased MPOD levels, along with increased serum levels of L, Z, and BDNF. These changes were associated with improved visual and cognitive performances and reduction in eye strain and eye fatigue in the children receiving LZ gummies. The investigational product was safe and well tolerated. TRIAL REGISTRATION: http://ctri.nic.in/ Identifier CTRI/2022/05/042364.

Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Study protocol for a 15-week randomised controlled trial assessing the independent effects of high-cholesterol and high-saturated fat diets on LDL cholesterol.

INTRODUCTION: Previous research has associated high dietary cholesterol intake with raised low-density lipoprotein cholesterol (LDL-C) and thus increased risk for cardiovascular disease (CVD). Emerging research suggests that it is saturated fat, not dietary cholesterol, associated with increased CVD risk. Despite being high in cholesterol, eggs, low in saturated fat, are not adversely associated with blood lipids or CVD risk. This paper describes a randomised controlled counter-balanced, cross-over trial assessing the effects of a high-cholesterol/low-saturated fat (egg) diet and a low-cholesterol/high-saturated fat diet (egg free) on blood lipids and lipoproteins, while accounting for physical activity levels which can also influence these parameters. The primary aim is to demonstrate that high cholesterol intake (from eggs) within a healthy, low-saturated fat diet does not adversely affect blood lipid levels and lipoprotein profiles. Instead, we propose that adverse effects on these parameters are mediated by saturated fat intake. The secondary aim is to explore relationships between changes in blood lutein and zeaxanthin concentrations and alterations in physical activity, examining whether changes in physical activity mediate effects on blood lipids and lipoproteins. METHODS AND ANALYSIS: Fifty-two adults aged 18-60 years with LDL-C less than 3.5 mmol/L will be randomly allocated to three isocaloric diets for 5 weeks each: a high-cholesterol (600 mg)/low-saturated fat (6%) (egg) diet, a low-cholesterol (300 mg)/high-saturated fat (12%) (egg free) diet and a control diet that is high in both cholesterol (600 mg) and saturated fat (12%). Lipid and lipoprotein levels, lipoprotein size and concentrations, blood pressure, blood glucose, physical activity levels, and plasma lutein and zeaxanthin concentrations will be measured. Treatment effects will be analysed using linear mixed effects models. ETHICS AND DISSEMINATION: Ethics approval was obtained from the University of South Australia Human Research Ethics Committee no. 204 327. Results will be disseminated through peer-reviewed journals and national and international presentations. TRIAL REGISTRATION NUMBER: NCT05267522.

The role of carotenoids in bone health-A narrative review.

Osteoporosis constitutes a serious challenge for public health. Dietary patterns belong to important, modifiable risk factors of this disease. Therefore, what and in what quantities we consume on a daily basis are extremely relevant. It is commonly known that bone health is positively affected by vitamins (e.g., vitamin D and vitamin K) as well as mineral components (e.g., calcium and magnesium). However, the human diet consists of many other groups of compounds that exhibit a potential antiosteoporotic and supporting bone-building effect. These dietary components include carotenoids. This paper presents a broad review of studies on the influence of particular carotenoids (ß-carotene, lutein, zeaxanthin, ß-cryptoxanthin, and lycopene) on bones. The paper discusses up-to-date in vitro experiments and research on animal models and presents how the results translate into clinical effect in humans.

Carotenoid degradation rate in milled grain of dent maize hybrids and its relationship with the grain physicochemical properties.

Carotenoids in maize grain degrade during storage, but the relationship between their stability and the physicochemical properties of the grain is unclear. Therefore, the carotenoid degradation rate in milled grain of three dent hybrids differing in grain hardness was evaluated at various temperatures (-20, 4 and 22 °C). The carotenoid degradation rate was calculated using first-order kinetics based on the content in the samples after 7, 14, 21, 28, 42, 56, 70 and 90 days of storage and related to the physicochemical properties of the grain. The highest grain hardness was found in the hybrid with the highest zein and endosperm lipid concentration, while the lowest grain hardness was found in the hybrid with the highest amylose content and the specific surface area of starch granule (SSA). As expected, carotenoids in milled maize grain were most stable at -20 °C, followed by storage at 4 and 22 °C. Tested hybrids differed in the degradation rate of zeaxanthin, α-cryptoxanthin and ß-carotene, and these responses were also temperature-dependent. In contrast, all hybrids showed similar degradation rate for lutein and ß-cryptoxanthin regardless of the storage temperature. Averaged over the hybrids, the degradation rate for individual carotenoids ranked as follows: lutein < zeaxanthin < α-cryptoxanthin < ß-cryptoxanthin < ß-carotene. The lower degradation rate for most carotenoids was mainly associated with a higher content of zein and specific endosperm lipids, with the exception of zeaxanthin, which showed an opposite pattern of response. Degradation rate for lutein and zeaxanthin negatively correlated with SSA, but interestingly, small starch granules were positively associated with higher degradation rate for mostcarotenoids. Dent-type hybrids may differ significantly in carotenoid degradation rate, which was associated with specific physicochemical properties of the maize grain.