Macular pigment-enriched oil production from genome-edited microalgae.

BACKGROUND: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. RESULTS: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g-1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g-1 DCW), and lipids (450.09 ± 25.48 mg g-1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g-1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g-1 lutein of oil and 7.69 ± 1.03 mg g-1 zeaxanthin of oil was produced. CONCLUSION: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

Effect of the emulsifier type on the physicochemical stability and in vitro digestibility of a lutein/zeaxanthin-enriched emulsion.

Lutein (L) and zeaxanthin (Z), as macular pigments, are water-insoluble, chemically unstable, and have low bioaccessibilities; they are often emulsified to overcome these limitations. This study investigated the impact of various emulsifiers (ethyl lauroyl arginate (LAE); Tween 80; and sodium dodecyl sulfate (SDS)) on the physicochemical properties and in vitro digestibilities of L/Z-fortified oil-in-water emulsions. Droplet aggregation and creaming extents were dependent on the emulsifier type. The ζ-potentials of emulsions stabilized by LAE, Tween 80, and SDS were + 87, - 26, and - 95 mV, respectively. SDS-stabilized emulsion had the smallest particles, while the particle sizes for the LAE- and Tween 80-stabilized emulsions were larger and not significantly different. The rates of L/Z degradation were sensitive to the emulsifier type and to heat, not to light. The L/Z bioaccessibility was the highest for the Tween 80 emulsion. Surfactants should therefore be carefully selected to optimize L/Z physicochemical stability and bioaccessibility in emulsions.

Formation of a novel coating material containing lutein and zeaxanthin via a Maillard reaction between bovine serum albumin and fucoidan.

The effective delivery of bioactive compounds has recently been receiving attention. In this study, a conjugate with BSA and fucoidan synthesized via the Maillard reaction was confirmed through electrophoresis, the o-phthalaldehyde assay, and through changes in absorbance. Two moles of fucoidan were glycated with one mole of BSA at 60 °C and 79% relative humidity for 4 days. The droplet coated with B-F conjugate remained stable during storage at 4 and 25 °C and slightly increased only at 55 °C however, the droplet coated with intact BSA and B/F mixture significantly increased. L/Z were degraded about 82, 79, and 36% for 4, 25, and 55 °C, respectively, regardless of the type of emulsifier. Although the conjugates could not prevent the degradation of lutein and zeaxanthin during storage, they improved the stability of the emulsion and showed 4.20-fold and 1.32-fold higher bioaccessibility than intact BSA and B/F mixtures, respectively.

Employment and the sexual and reproductive behavior of female adolescents.

CONTEXT: Women's employment opportunities may reduce the risk of early intercourse and pregnancy, but some evidence has linked adolescent employment and problem behaviors with early intercourse. METHODS: Hazard regression analyses of data from the National Longitudinal Survey of Youth were used to examine the relationship between employment and the risk of first intercourse before age 20 among women who were aged 14-16 in 1979. The relationship between employment and the risk of a first, nonmarital pregnancy among sexually experienced young women was also assessed. RESULTS: Current employment and cumulative months of past employment are associated with increased hazards of first intercourse (hazard ratios, 1.20 and 1.01, respectively); this association is particularly strong for white young women. Adolescents who work more than 120 hours a month are significantly more likely than nonworking adolescents to experience first intercourse (1.4). Although current employment has no effect on the likelihood of a first, nonmarital pregnancy among white adolescents, it is associated with an increased risk of pregnancy among blacks and with a reduced risk of pregnancy among Hispanics. CONCLUSIONS: Program planners and policymakers should be aware of the potential association between adolescent employment, particularly intense employment, and the likelihood of initiating intercourse and experiencing pregnancy, even if causality is still unclear.