Engineered Maize Hybrids with Diverse Carotenoid Profiles and Potential Applications in Animal Feeding.

Multi-gene transformation methods need to be able to introduce multiple transgenes into plants in order to reconstitute a transgenic locus where the introduced genes express in a coordinated manner and do not segregate in subsequent generations. This simultaneous multiple gene transfer enables the study and modulation of the entire metabolic pathways and the elucidation of complex genetic control circuits and regulatory hierarchies. We used combinatorial nuclear transformation to produce multiplex-transgenic maize plants. In proof of principle experiments, we co-expressed five carotenogenic genes in maize endosperm. The resulting combinatorial transgenic maize plant population, equivalent to a "mutant series," allowed us to identify and complement rate-limiting steps in the extended endosperm carotenoid pathway and to recover corn plants with extraordinary levels of ß-carotene and other nutritionally important carotenoids. We then introgressed the induced (transgenic) carotenoid pathway in a transgenic line accumulating high levels of nutritionally important carotenoids into a wild-type yellow-endosperm variety with a high ß:ε ratio. Novel hybrids accumulated zeaxanthin at unprecedented amounts. We introgressed the same pathway into a different yellow corn line with a low ß:ε ratio. The resulting hybrids, in this case, had a very different carotenoid profile. The role of genetic background in determining carotenoid profiles in corn was elucidated, and further rate-limiting steps in the pathway were identified and resolved in hybrids. Astaxanthin accumulation was engineered by overexpression of a ß-carotene ketolase in maize endosperm. In early experiments, limited astaxanthin accumulation in transgenic maize plants was attributed to a bottleneck in the conversion of adonixanthin (4-ketozeaxanthin) to astaxanthin. More recent experiments showed that a synthetic ß-carotene ketolase with a superior ß-carotene/zeaxanthin ketolase activity is critical for the high-yield production of astaxanthin in maize endosperm. Engineered lines were used in animal feeding experiments which demonstrated not only the safety of the engineered lines but also their efficacy in a range of different animal production applications.

Provitamin A carotenoids from an engineered high-carotenoid maize are bioavailable and zeaxanthin does not compromise ß-carotene absorption in poultry.

High-carotenoid (HC) maize, a biofortified staple crop which accumulates ß-carotene, ß-cryptoxanthin, lutein and zeaxanthin, was used as a feed component in a chicken feeding trial to assess the bioavailability of provitamin A (PVA) carotenoids in the kernel matrix compared to the synthetic and natural color additives routinely used in the poultry industry. We found that the PVA carotenoids in HC maize were not metabolized in the same manner: ß-carotene was preferentially converted into retinol in the intestine whereas ß-cryptoxanthin accumulated in the liver. We also considered the effect of zeaxanthin on the absorption of PVA carotenoids because zeaxanthin is the major carotenoid component of HC maize. We found that chickens fed on diets with low levels of zeaxanthin accumulated higher levels of retinol in the liver, suggesting that zeaxanthin might interfere with the absorption of ß-carotene, although this observation was not statistically significant. Our results show that HC maize provides bioavailable carotenoids, including PVA carotenoids, and is suitable for use as a feed component.

Influence of Cooking Conditions on Carotenoid Content and Stability in Porridges Prepared from High-Carotenoid Maize.

Maize is a staple food crop in many developing countries, hence becoming an attractive target for biofortification programs toward populations at risk of micronutrient deficiencies. A South African white endosperm maize inbred line was engineered with a carotenogenic mini-pathway to generate high-carotenoid maize, which accumulates ß-carotene, lutein and zeaxanthin. As maize porridge is a traditional meal for poor populations in sub-Saharan African countries, high-carotenoid maize was used as raw material to prepare different maize meals. The objective of this work was to assess the impact of popular home-cooking techniques and different cooking parameters (temperature, time and pH) on the final carotenoid content in the cooked product, using a spectrophotometric technique based on the mean absorption of carotenoids at 450 nm. Carotenoid levels were not only preserved, but also enhanced in high-carotenoid maize porridges. The carotenoid content was increased when temperatures ≤95 °C were combined with short cooking times (10-60 min). The most optimum thermal treatment was 75 °C/10 min. When treated under those conditions at pH 5, high-carotenoid maize porridges doubled the initial carotenoid content up to 88 µg/g dry weight. Regarding to cooking techniques, the highest carotenoid content was found when unfermented thin porridges were prepared (51 µg/g dry weight of high-carotenoid maize porridge). We conclude that high-carotenoid maize may contribute to enhance the dietary status of rural populations who depend on maize as a staple food.

A Combination of Apple Vinegar Drink with Bacillus coagulans Ameliorates High Fat Diet-Induced Body Weight Gain, Insulin Resistance and Hepatic Steatosis.

Obesity is a worldwide epidemic characterized by excessive fat accumulation, associated with multiple comorbidities and complications. Emerging evidence points to gut microbiome as a driving force in the pathogenesis of obesity. Vinegar intake, a traditional remedy source of exogenous acetate, has been shown to improve glycemic control and to have anti-obesity effects. New functional foods may be developed by supplementing traditional food with probiotics. B. coagulans is a suitable choice because of its resistance to high temperatures. To analyze the possible synergic effect of Vinegar and B. coagulans against the metabolic alterations induced by a high fat diet (HFD), we fed twelve-week-old C57BL/6 mice with HFD for 5 weeks after 2 weeks of acclimation on a normal diet. Then, food intake, body weight, blood biochemical parameters, histology and liver inflammatory markers were analyzed. Although vinegar drink, either alone or supplemented with B. coagulans, reduced food intake, attenuated body weight gain and enhanced glucose tolerance, only the supplemented drink improved the lipid serum profile and prevented hepatic HFD-induced overexpression of CD36, IL-1ß, IL-6, LXR and SREBP, thus reducing lipid deposition in the liver. The beneficial properties of the B. coagulans-supplemented vinegar appear to be mediated by a reduction in insulin and leptin circulating levels.

Biofortification of crops with nutrients: factors affecting utilization and storage.

Biofortification is an effective and economical method to improve the micronutrient content of crops, particularly staples that sustain human populations in developing countries. Whereas conventional fortification requires artificial additives, biofortification involves the synthesis or accumulation of nutrients by plants at source. Little is known about the relative merits of biofortification and artificial fortification in terms of nutrient bioaccessibility and bioavailability, and much depends on the biochemical nature of the nutrient, which can promote or delay uptake, and determine how efficiently different nutrients are transported through the blood, stored, and utilized. Data from the first plants biofortified with minerals and vitamins provide evidence that the way in which nutrients are presented can affect how they are processed and utilized in the human body. The latest studies on the effects of the food matrix, processing and storage on nutrient transfer from biofortified crops are reviewed, as well as current knowledge about nutrient absorption and utilization.

The distribution of carotenoids in hens fed on biofortified maize is influenced by feed composition, absorption, resource allocation and storage.

Carotenoids are important dietary nutrients with health-promoting effects. The biofortification of staple foods with carotenoids provides an efficient delivery strategy but little is known about the fate and distribution of carotenoids supplied in this manner. The chicken provides a good model of human carotenoid metabolism so we supplemented the diets of laying hens using two biofortified maize varieties with distinct carotenoid profiles and compared the fate of the different carotenoids in terms of distribution in the feed, the hen's livers and the eggs. We found that after a period of depletion, pro-vitamin A (PVA) carotenoids were preferentially diverted to the liver and relatively depleted in the eggs, whereas other carotenoids were transported to the eggs even when the liver remained depleted. When retinol was included in the diet, it accumulated more in the eggs than the livers, whereas PVA carotenoids showed the opposite profile. Our data suggest that a transport nexus from the intestinal lumen to the eggs introduces bottlenecks that cause chemically-distinct classes of carotenoids to be partitioned in different ways. This nexus model will allow us to optimize animal feed and human diets to ensure that the health benefits of carotenoids are delivered in the most effective manner.