Stage I

 Preliminary analysis and documentation for the development of feed additives rich in carotenoids

We conducted a thorough study to present the identified problem and the solutions to be implemented through this project. The first chapter, overview, gives general data on carotenoids on the whole and on xanthophylls, in particular. The chapter shows studies and data regarding: (i) the general properties of the carotenoids; (ii) chemistry of lutein and xanthophyll and their role in preserving human health; (iii) importance of the hen egg as source of xanthophyll for humans.
The second chapter makes reference to the European norms and to the limits imposed to the use of feed additives rich in carotenoids/xanthophyll in poultry feeding. We show: (i) the sources of lutein and zeaxanthin used in poultry feeding and their efficiency in colouring the hen eggs; (ii) evaluation of the risks/maximum amount of xanthophylls admitted in compound feeds in agreement with the scientific advices of EFSA (European Food Safety Authority).
Chapter three includes technical documentation about the feed additives rich in carotenoids, which can be tested during the project. The documentation refers to (i) the feeding and commercial value of the carotenoids-rich feed additives; (ii) documentation on the extraction, separation and determination of the carotenoids from plant extracts; (iii) presentation of raw plant material selected according to their content of carotenoids, yellow-orange coloured flavones and other essential nutrients, which will be studied in terms of their efficiency to pigment the egg.
Chapter four presents the chemical methods developed and validated for the determination of lutein and xanthine from plant materials and from egg yolk. This chapter includes: (i) review of the methods described by the literature; (ii) presentation of two methods that were developed for lutein and zeaxanthin determination from feeds (except from cereal grains) and eggs; (iii) presentation of two specific testing procedures (PIS) in the format set by the Quality Manual of the Laboratory of chemistry and nutrition physiology from IBNA; (iv) presentation of the “in house” validation of the liquid chromatographic method for lutein and zeaxanthin determination by HPLC. Validation is presented in a Validation Report.
The study ends with conclusions and a selective list of references.

Conclusions resulted from the activities of this stage:

  • During the current period when, nationally and internationally, the focus is on the better use of the grown and wild plants, of the natural resources in general, according to their chemical composition, the knowledge of biologically active substances is fundamental for the production of functional foods. Only by knowing the chemical composition of the plants, the biochemical mechanisms underlying the biological phenomena, one may finally develop poultry feeds high in biologically active matter, which enhance the biological value of the egg yolk by a higher supply of lutein and zeaxanthin.
  • Lutein and its stereoisomer, zeaxanthin, belong to the xanthophyll family of the carotenoids. Unlike the hydrocarbon carotenoids, such as β-carotene and lycopene, lutein and zeaxanthin have two hydroxyl groups, one on each side of the molecule. These groups are thought to play an essential role in the biological function of lutein and zeaxanthin and in the identification of the adequate chemical methods to determine these xanthophylls.
  • Lutein (3R, 3’R, 6’R) – β, ε-carotene-3,3′-diol), CAS number 127-40-2 (corresponding number in CE database is 204 – 840 – 0), has the molecular formula C40H56O2 and the molar mass 568.88 g /mol. Zeaxanthin all-trans-(3R, 3’R)- β – carotene-3,3’–diol), CAS number 144-68-3, has the same chemical formula (C40H56O2) and a molecular weight of 568.9 g /mol. Lutein and zeaxanthin have identical chemical formulas and are isomers, but not stereoisomers. The only difference between these two xanthophylls is the location of the double bond in one of the terminal rings.
  • The uniqueness of lutein and zeaxanthin lies in their presence in eye tissues. Lutein and zeaxanthin are highly concentrated in the macula, a small portion of the retina that is responsible for the central vision and for visual acuity, being the only carotenoids from this tissue. Lutein and zeaxanthin are the only carotenoids present in the crystalline lens, another eye tissue that is essential for eyesight.
  • Presently, macular degeneration (MD) is the main cause for blindness in the developed countries. The disease is characterized by progressive, degenerative and irreversible alteration of the central retina area (macula). Macula is the area from the eye retina responsible for sharp-sightedness. It is yellowish due to a yellow pigment. Macular degeneration develops progressively and it affects over 5% of the people over 65. The dry form of DM is receptive to the administration of nutrients such as vitamins and minerals. Of the 600 known carotenoids, lutein and zeaxanthin are the major carotenoids from the eye retina macula in humans.
  • The supply of lutein in the diets of the European consumers is about 2.2 mg/day, while in the diets of the American consumers is about 1.7 mg/day. This is due to the decrease of the consumption of vegetables with green leaves. The scientific data show that a daily intake of 6-14 mg lutein may be associated to a 50% reduction of the risk for age-related macular degeneration and cataract.
  • Different studies have shown that the consumption of foods/food supplements rich in xanthophylls increase the optic density of the macular pigment (indirect measurement of the lutein concentration) and may help improving the visual function of the patients with age-related macular degeneration, cataract or other eye dysfunctions.
  • The positive effects of lutein and zeaxanthin on the eye are just part of the benefits for their consumers. There are studies documenting that lutein and zeaxanthin are also beneficial to heart health by reducing the risk of arteriosclerosis; they reduce the development of disorders that endanger the immunological status; they may inhibit cell proliferation, cell transformation and may modulate the expression of the genetic determinants in the prevention of certain types of cancer.
  • The dark-green fruits and vegetables are important sources of lutein and zeaxanthin. Their consumption is associated to higher concentrations of macular pigment. The egg yolk too, a matrix of digestible lipids, cholesterol, triglycerides, phospholipids, also contains xanthophylls (lutein and zeaxanthin) dispersed within them matrix next to other liposoluble nutrients such as liposoluble vitamins. Lutein and zeaxanthin from the lipid matrix of the egg yolk have a higher bioavailability than in the vegetable sources, as shown by recent studies.
  • The concentration of lutein and zeaxanthin from the eggs can vary considerable, depending on the type of feed given to the layers and on the conditions of rearing. The laying hens reared on the floor produce eggs with twice as much carotenoids in the yolk than the laying hens reared in battery cages.
  • The lutein from plants is usually found as ester, while the lutein from the egg is in its free form. It has been thought until not so long ago that lutein bioavailability is not influenced so much by esterification, as by its solubilisation in the matrix of the food. Recent studies have shown, however, that the serum lutein concentrations obtained after the administration of free lutein were 20% higher than those obtained after the administration of esterified lutein.
  • Egg yolk and broiler skin pigmentation is usually done using feeds that have high levels of lutein and zeaxanthin, supplemented with other sources of natural or synthetic carotenoids.
  • The raw materials rich in lutein include: alfalfa meal, yellow corn, red corn and corn gluten. The by-products from the food industry can be used to diversify the range of feed additives/ingredients.
  • The European legislation set the highest allowed individual content of carotenoids and xanthophylls to 80 mg/ kg feed ingredient.
  • Lutein exists both in feed ingredients and in feed additives in association with zeaxanthin in various ratios (1:0.1 to 0.8). Therefore, it is not possible to evaluate individually the pigmenting efficiency of lutein, particularly because zeaxanthin produces a much more intense colour than lutein. Therefore, lutein supplementation is rarely calculated as such, but rather as part of the total xanthophylls.
  • There is little information on animal tolerance to carotenoids/xanthophylls. However, given the natural incidence of carotenoids/xanthophylls approved as dyestuffs and taking into account the structural similarity of these substances, FEEDAP Commission sees no reasons to worry.
  • Partner 2 (SC Hofigal SA) identified several indigenous plants and by-products from the industry of food supplements that fit the purpose of the project (development of eggs enriched in lutein and zeaxanthin), which will be studied to determine their efficacy as natural pigments for laying hens diets: French marigold flowers (Tagetes patula); pot marigold flowers (Calendula officinalis); pumpkin meal (Curcubita maxima); buckthorn fruits meal (Hippophae rhamnoides); hemp seeds meal (Cannabis sativum); alfalfa meal (Medicago sativa); dry tomato skins (Licopersicum esculentum); cabbage (Brassica oleracea); carrot (Daucus Carota).
  • The trends in carotenoid analysis reflect not just the improvement of the analytical instruments, but also the perception on the broader role of these compounds, from dyeing to protection against the degenerative disorders.
  • The main problem with the assay of carotenoids comes from their instability. Thus, irrespective of the analytical method, precautions need to be taken to avoid the formation or artefacts and the loss of quantity. These measures include the performance of analysis as fast as possible, exclusion of oxygen, protection against light, avoiding high temperatures, avoiding the contact with acids and using highly pure solvents which are free of deleterious impurities (Schiedt and Liaaen-Jensen, 1995).During this stage, the collective of the liquid chromatography of the Laboratory for chemistry and physiology nutrition within IBNA Balotesti (P1) developed 2 methods for lutein and zeaxanthin determination from feeds (except from cereal grains) and from egg yolk. These new methods were transcripted in the format of the specific testing procedures stipulated by Quality Manual of the Laboratory of chemistry and nutrition physiology from IBNA. We also did the “in house” validation of the liquid chromatographic method for lutein and zeaxanthin determination by HPLC. The analytical method was verified using certified reference material certified by BCR (Community Bureau of Reference, European Commission) and reviewed by IRMM (Institute of Reference Materials and Measurements). This material contains 25 g lyophilised vegetable material (preserved tomatoes, frozen carrots and sweet potatoes). The certified values are expressed in mg/kg dry matter. The certificate recommends using reverse-phase HPLC and detection with UV/Vis spectrophotometer or with diode Array. In order to make the correction for dry matter, we determined the dry matter on the same day when lutein and zeaxanthin were determined