Spectrophotometric determination of yellow pigment content and evaluation of carotenoids by high-performance liquid chromatography in durum wheat grain

The so-called "yellow pigment" content of durum wheat has been used for a long time as an indicator of the color quality of durum wheat and pasta products. For decades the chemical nature of these pigments has been assigned to carotenoids, mainly to the xanthophyll lutein and its fatty acid esters. The chemical composition of the yellow pigments of eight German durum wheat cultivars was studied. Grains were milled on a laboratory mill. Pigment extraction of millstream fractions was performed according to the optimized ICC standard method 152 procedure, and the chemical composition of the extract was analyzed by isocratic reversed phase high-performance liquid chromatography. all-trans-Lutein ranged from 1.5 to 4 mg kg(-1), and zeaxanthin was found in traces. No lutein esters and carotenes were detected. Surprisingly, the fraction of carotenoids of the complete yellow pigment content amounted to only 30-50% of the yellow pigment quantities, so there are still compounds in durum wheat not yet identified that contribute considerably to the yellow color of the grain extracts. The isolation and chemical identification of those pigments are under investigation.     

Normal phase high-performance liquid chromatography method for the determination of tocopherols and tocotrienols in cereals

The eight vitamers of vitamin E (alpha-, beta-, gamma-, and delta-tocopherols and -tocotrienols) have different antioxidant and biological activities and have different distributions in foods. Some cereals, especially oat, rye, and barley, are good sources of tocotrienols. A fast procedure for the determination of tocopherols and tocotrienols (tocols) in cereal foods was developed. It involves sample saponification and extraction followed by normal phase high-performance liquid chromatography (HPLC). The results have been compared with those found by direct extraction without saponification. The method is sensitive and selective enough to be tested on a wide variety of cereal samples. The highest tocol levels were found in soft wheat and barley ( approximately 75 mg/kg of dry weight). beta-Tocotrienol is the main vitamer found in hulled and dehulled wheats (from 33 to 43 mg/kg of dry weight), gamma-tocopherol predominates in maize (45 mg/kg of dry weight) ), and alpha-tocotrienol predominates in oat and barley (56 and 40 mg/kg of dry weight, respectively).     

Estimation of color of durum wheat. Comparison of WSB, HPLC, and reflectance colorimeter measurements

Color is an important parameter involved in the definition of semolina and pasta quality. This character is mainly due to natural pigments (carotenoids) that are present at different levels in cereals and cereal products, due to botanical origin, growing conditions, distribution in the kernel, and technological processes. In food industries, color measurements are usually performed by means of automatic instruments that are rapid and safe, as alternatives to the chemical extraction methods. In this study, automatic measurements (CIE, color-space system L, a, b), water-saturated butanol (WSB), and HPLC determinations have been applied to evaluate the carotenoid content in whole meals and respective semolina samples produced from wheat cultivated in the years 2001 and 2002. In whole meals, total carotenoids, determined by HPLC, were about 3.0 microg/g (2001) and 3.5 microg/g (2002) calculated on dry weight (dw) and about 3.0 and 3.2 microg/g dw in corresponding semolina samples. The b values for the same period were 19.78 and 15.75, respectively, in raw materials and 20.03-21.67 in semolina. Results have confirmed lutein and beta-carotene as the main components mainly responsible for the yellow color in wheat grains. The ability of the index b to express natural dyeing was dependent on sample characteristics as demonstrated by the relationships found between this index and pigments, although the best correlation resulted between HPLC and WSB.     

Determination of major carotenoids in a few Indian leafy vegetables by high-performance liquid chromatography

Leafy vegetables [Basella rubra L., Peucedanum sowa Roxb., Moringa oleifera Lam., Trigonella foenum-graecum L., Spinacia oleracea L., Sesbania grandiflora (L.) Poir., and Raphanus sativus L.] that are commonly used by the rural population in India were evaluated in terms of their main carotenoid pattern. The extracted carotenoids were purified by open column chromatography (OCC) on a neutral alumina column to verify their identity by their characteristic UV-visible absorption spectra. Reverse-phase high-performance liquid chromatography (HPLC) on a C18 column with UV-visible photodiode array detection under isocratic conditions was used for quantification of isolated carotenoids. Acetonitrile/methanol/dichloromethane (60:20:20 v/v/v) containing 0.1% ammonium acetate was used as a mobile phase. The major carotenoids identified by both methods were lutein, beta-carotene, violaxanthin, neoxanthin, and zeaxanthin. Among the carotenoids identified, lutein and beta-carotene levels were found to be higher in these leafy vegetables. Results show that P. sowa and S. oleracea are rich sources of lutein (77-92 mg/100 g of dry wt) and beta-carotene (36-44 mg/100 g of dry wt) compared with other leafy vegetables. The purity of carotenoids eluted by OCC was clarified by HPLC, and they were found to be 92% +/- 3% for neoxanthin, 94% +/- 2% for violaxanthin, 97% +/-2% for lutein and zeaxanthin, and 90% +/- 3% for beta-carotene. It could be recommended to use P. sowa and S. oleracea as rich sources of lutein and beta-carotene for health benefits. The OCC method proposed is relatively simple and provides purified carotenoids for feeding trials.     

Carotenoid pigments in Rosa mosqueta hips, an alternative carotenoid source for foods

Carotenoid composition has been investigated in Rosa mosqueta hips (Rosa rubiginosa, Rosa eglanteria). Six major carotenoids were identified (beta-carotene, lycopene, rubixanthin, gazaniaxanthin, beta-cryptoxanthin, and zeaxanthin) together with other minor carotenoids (violaxanthin, antheraxanthin, and gamma-carotene). An average composition has been estimated as follows: beta-carotene (497.6 mg/kg of dry wt), lycopene (391.9 mg/kg of dry wt), rubixanthin (703.7 mg/kg of dry wt), gazaniaxanthin (289.2 mg/kg of dry wt), beta-cryptoxanthin (183.5 mg/kg of dry wt), zeaxanthin (266. 6 mg/kg of dry wt), and minor carotenoids (67.1 mg/kg of dry wt). Possible uses in food technology are outlined and discussed including the preparation of highly colored oleoresins as natural colorants of food and beverages and as provitamin A sources.     

Evaluation of analytical methods for carotenoid extraction from biofortified maize (Zea mays sp.)

Biofortification of maize with beta-carotene has the potential to improve vitamin A status in vitamin A deficient populations where maize is a staple crop. Accurate assessment of provitamin A carotenoids in maize must be performed to direct breeding efforts. The objective was to evaluate carotenoid extraction methods and determine essential steps for use in countries growing biofortified maize. The most reproducible method based on coefficient of variation and extraction efficiency was a modification of Kurilich and Juvik (1999). Heat and saponification are required to release carotenoids from biofortified maize and remove oils interfering with chromatographic analysis. For maize samples with high oil content, additional base may be added to ensure complete saponification without compromising results. Degradation of internal standard before carotenoids were released from the maize matrix required the addition of internal standard after heating to prevent overestimation of carotenoids. This modified method works well for lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, and beta-carotene.     

Kinetics of carotenoids degradation during the storage of einkorn (Triticum monococcum L. ssp. monococcum) and bread wheat (Triticum aestivum L. ssp. aestivum) flours

To evaluate the effect of storage temperature, the degradation kinetics of carotenoids in wholemeal and white flour of einkorn cv. Monlis and bread wheat cv. Serio, stored at -20, 5, 20, 30, and 38 degrees C, was assessed by normal-phase high-performance liquid chromatography. In Monlis, the carotenoids content (8.1 and 9.8 mg/kg for wholemeal and white flour, respectively) was 8-fold higher than in Serio (1.0 and 1.1 mg/kg). Only lutein and zeaxanthin were detected in bread wheat, while significant quantities of (alpha and beta)-carotene and beta-cryptoxanthin were observed in einkorn. Carotenoids degradation was influenced by temperature and time, following first-order kinetics. The degradation rate was similar in wholemeal and white flour; however, loss of lutein and total carotenoids was faster in Serio than in Monlis. The activation energy E(a) ranged from 35.2 to 52.5 kJ/mol. Temperatures not exceeding 20 degrees C better preserve carotenoids content and are recommended for long-term storage.     

Carotenoid bioaccessibility from whole grain and degermed maize meal products

Although yellow maize (Zea mays) fractions and products are a source of dietary carotenoids, only limited information is available on the bioavailability of these pigments from maize-based foods. To better understand the distribution and bioavailability of carotenoid pigments from yellow maize (Z. mays) products, commercial milled maize fractions were screened for carotenoid content as were model foods including extruded puff, bread, and wet cooked porridge. Carotenoid content of maize fractions ranged from a low of 1.77-6.50 mg/kg in yellow maize bran (YCB) to 12.04-17.94 mg/kg in yellow corn meal (YCM). Lutein and zeaxanthin were major carotenoid species in maize milled fractions, accounting for approximately 70% of total carotenoid content. Following screening, carotenoid bioaccessibility was assessed from model foods using a simulated three-stage in vitro digestion process designed to measure transfer of carotenoids from the food matrix to bile salt lipid micelles (micellarization). Micellarization efficiency of xanthophylls was similar from YCM extruded puff and bread (63 and 69%), but lower from YCM porridge (48%). Xanthophyll micellarization from whole yellow corn meal (WYCM) products was highest in bread (85%) and similar in extruded puff and porridge (46 and 47%). For extruded puffs and breads, beta-carotene micellarization was 10-23%, but higher in porridge (40-63%), indicating that wet cooking may positively influence bioaccessibility of apolar carotenes. The results suggest that maize-based food products are good dietary sources of bioaccessible carotenoids and that specific food preparation methods may influence the relative bioaccessibility of individual carotenoid species.     

Comparison of nutritional traits variability in selected eighty-seven inbreds from Chinese maize (Zea mays L.) germplasm

Among cereals, only maize has not only a high amount of carotenoids, tocopherols, and oil content but also is rich in starch and protein content compared with other major food crops, such as rice and wheat. The present investigation was made primarily to assess the genetic variability for nutritionally important traits in 87 elite maize inbreds representing major heterotic groups in China. Carotenoid and tocopherol fractions were measured by high-performance liquid chromatography (HPLC), whereas oil, starch, and protein contents were detected by a VECTER22/N near-infrared analyzer. Significant interactions between genotypes and years were observed for all the traits. The pooled mean values of beta-carotene, beta-cryptoxanthin, alpha-carotene, lutein, zeaxanthin, and total carotenoids were 0.449, 0.876, 0.121, 5.803, 3.048, and 10.298 microg g (-1), respectively, whereas the combined mean performance of alpha-tocopherol, gamma-tocopherol, delta-tocopherol, and total tocopherols were 23.98, 32.90, 2.189, and 59.55 microg g (-1), respectively. The average protein, starch, and oil contents were observed to be 12.28, 64.51, and 3.55%, respectively. High level of heritability estimates were observed for all the traits and ranged from 65.6% (protein content) to 92.5% (alpha/gamma-tocopherol ratio). Most of the traits studied in this experiment were either significantly positive correlated or independent. The present finding exhibits substantial opportunities to the breeders for improvement of these traits in maize cultivars and also suggests further exploration of a new source of elite breeding stocks containing a high level of these nutritionally important compounds. Finally, these findings may also help in biofortification of maize.     

Determination of beta-carotene and lutein available from green leafy vegetables by an in vitro digestion and colonic fermentation method

Green leafy vegetables (Spinacea oleracea, Cnidoscolus aconitifolius, and Solanum americanum) contain a high amount of beta-carotene (27-52 mg/100 g of dry sample) and lutein (140-193 mg/100 g of dry sample). The amount of beta-carotene and lutein released from the food matrix by the action of digestive enzymes ranged from 22 to 67% and from 27 to 77%, respectively. There was a significant correlation between the enzymatic release of carotenoids (lutein + beta-carotene) and the content of Klason lignin, nonstarch polysaccharides, and resistant protein. The carotenoids released by the in vitro colonic fermentation ranged from 2 to 11%, and part of them (0.251-4.03 mg/100 g of original dry sample) remained intact in the fermentation media and could be potentially absorbed in the colon. A significant part of carotenoids seems to be unavailable in the intestinal tract (16% in S. oleracea to 58% in C. aconitifolius).     

Identification and quantification of seed carotenoids in selected wheat species

Selected primitive and modern wheat species were evaluated on the basis of their carotenoid composition and effects of the genotype and environment on lutein using spectrometry and liquid chromatography. Carotenoids in the wheat extracts were identified and confirmed on the basis of their UV/vis and mass spectra compared with those of authentic standards. The protonated molecule (M + 1)+ at m/z 569 was the predominant ion for zeaxanthin compared to the fragment ion at m/z 551 for lutein. A similar carotenoid profile was obtained for the wheat species investigated, but significant differences were observed in the concentration of carotenoids. Einkorn (Triticum monococcum) exhibited the highest level of all-trans-lutein, averaging 7.41 microg/g with small amounts of all-trans-zeaxanthin, cis-lutein isomers, and beta-carotene. Durum, Kamut, and Khorasan (Triticum turgidum) had intermediate levels of lutein (5.41-5.77 microg/g), while common bread or pastry wheat (Triticum aestivum) had the lowest content (2.01-2.11 microg/g). Lutein in einkorn appeared to be influenced significantly by environmental growing conditions.     

Quantification of carotenoid and tocopherol antioxidants in Zea mays.

Recent investigations into carotenoid and tocopherol biological activity in mammalian systems indicate that these antioxidants are associated with the prevention of degenerative diseases. Both carotenoids and tocopherols can be found in corn kernel tissue. A replicated survey of 44 sweet and dent corn lines was conducted to determine qualitative and quantitative variability of lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, and beta-carotene, as well as the alpha-, delta-, and gamma- forms of tocopherol. The primary carotenoids in fresh market sweet corn were found to be lutein and zeaxanthin, with the gamma form dominating among the tocopherols. Mean values among the genotypes were observed to range from 0 to 20.0 and 2.4 to 63.3 microg/g dry weight for lutein and gamma-tocopherol, respectively, indicating variability among genotypes in genes regulating the metabolism of these compounds. The observed genetic variability suggests profound differences in potential health promotion among genotypes and supports the feasibility of developing germplasm with enhanced levels of these antioxidant compounds at dosages that could promote health among the consuming public.     

The xanthophyll composition of biofortified maize (Zea mays Sp.) does not influence the bioefficacy of provitamin a carotenoids in Mongolian gerbils (Meriones unguiculatus)

Maize has been targeted for biofortification with provitamin A carotenoids through traditional breeding. Two studies were conducted in gerbils to evaluate factors that may affect provitamin A activity. Maize diets had equal theoretical concentrations of vitamin A (VA) assuming 100% bioefficacy. Study 1 ( n = 57) varied the ratio of beta-cryptoxanthin and beta-carotene but maintained the same theoretical VA. Study 2 ( n = 67) varied lutein and zeaxanthin. Other treatments were oil, VA, or beta-carotene doses. Serum and livers were analyzed for VA and carotenoids. In study 1, total liver VA did not differ among the maize groups. In study 2, total liver VA of the VA and maize groups were higher than controls ( P < 0.05). Conversion factors were 2.1-3.3 mug beta-carotene equivalents to 1 mug retinol. Twice the molar amount of beta-cryptoxanthin was as efficacious as beta-carotene and the proportion of beta-cryptoxanthin or xanthophylls did not appreciably change the VA value of biofortified maize.     

Alkylresorcinols in cereals and cereal products

The alkylresorcinol (AR) content of 8 commonly consumed cereals, 125 Triticum cultivars, milling fractions of wheat and rye, bread, and other cereal products was analyzed. ARs were found in wheat (489-1429 microgram/g), rye (720-761 microgram/g), triticale (439-647 microgram/g), and barley (42-51 microgram/g), but not in rice, oats, maize, sorghum, or millet. One durum wheat variety was found to have an exceptionally low level of ARs (54 microgram/g) compared to other durum wheat varieties (589-751 microgram/g) and Triticumspecies analyzed. The AR content of milling fractions closely followed the ash content and could be used as a marker of the presence of bran in flour. Using hot 1-propanol extraction, all ARs could be extracted from bread, contrary to previous studies which suggested that ARs were destroyed during baking. Cereal products varied greatly in AR content, with those containing wheat bran or whole rye having the highest content.     

Carotenoid Composition and Changes in Sweet and Field Corn (Zea mays) During Kernel Development

Kernels of two carotenoid‐rich cultivars, sweet corn Jingtian 5 and field corn Suyu 29, were compared in terms of carotenoid composition during corn kernel development. The results showed that eight principal carotenoids were characterized by HPLC with diode array detection and atmospheric pressure chemical ionization tandem mass spectrometry with a C30 column. During kernel development, there was a similar trend in the change of total carotenoids for both corn cultivars, and the variation of individual carotenoids was also somewhat similar; violaxanthin, zeaxanthin, lutein, α‐cryptoxanthin, and β‐cryptoxanthin contents had upward trends, whereas neoxanthin content declined all the time, and α‐carotene and β‐carotene had no significant changes. However, the highest levels of the major carotenoids lutein (41.61 µg/g, dry weight) and zeaxanthin (39.59 µg/g, dry weight) obtained in field corn Suyu 29 during the milk stage were higher than those in sweet corn Jingtian 5, whereas the other individual carotenoid levels were significantly lower. Compared with the grain color, highly significant positive correlations were observed between zeaxanthin, lutein, and violaxanthin contents and deeper yellow/orange coloration indicators for field corn Suyu 29, but these relationships were weak for sweet corn Jingtian 5. Potential genetic variation might exist for carotenoid accumulation in sweet and field corn kernels.     

Distribution of lutein,zeaxanthin, and related geometrical isomers in fruit,vegetables, wheat,and pasta products

Quantitative data with regard to dietary (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, and their (E/Z)-geometrical isomers are scarce, and in most cases, only the combined concentrations of these two carotenoids in foods are reported. Lutein and zeaxanthin accumulate in the human macula and have been implicated in the prevention of age-related macular degeneration (AMD). The qualitative and quantitative distributions of lutein, zeaxanthin, and their (E/Z)-isomers in the extracts from some of the most commonly consumed fruits, vegetables, and pasta products were determined by HPLC employing a silica-based nitrile-bonded column. Green vegetables had the highest concentration of lutein (L) and zeaxanthin (Z), and the ratios of these carotenoids (L/Z) were in the range 12-63. The yellow-orange fruits and vegetables, with the exception of squash (butternut variety), had much lower levels of lutein in comparison to greens but contained a higher concentration of zeaxanthin. The ratio of lutein to zeaxanthin (L/Z) in two North American bread varieties of wheat (Pioneer, Catoctin) was 11 and 7.6, respectively, while in a green-harvested wheat (Freekeh) imported from Australia, the ratio was 2.5. Between the two pasta products examined, lasagne and egg noodles, the latter had a much higher concentration of lutein and zeaxanthin. The levels of the (E/Z)-geometrical isomers of lutein and zeaxanthin in these foods were also determined.     

Oxalate content of cereals and cereal products

Detailed knowledge of food oxalate content is of essential importance for dietary treatment of recurrent calcium oxalate urolithiasis. Dietary oxalate can contribute considerably to the amount of urinary oxalate excretion. Because cereal foods play an important role in daily nutrition, the soluble and total oxalate contents of various types of cereal grains, milling products, bread, pastries, and pasta were analyzed using an HPLC-enzyme-reactor method. A high total oxalate content (>50 mg/100 g) was found in whole grain wheat species Triticum durum (76.6 mg/100 g), Triticum sativum (71.2 mg/100 g), and Triticum aestivum (53.3 mg/100 g). Total oxalate content was comparably high in whole grain products of T. aestivum, that is, wheat flakes and flour, as well as in whole grain products of T. durum, that is, couscous, bulgur, and pasta. The highest oxalate content was demonstrated for wheat bran (457.4 mg/100 g). The higher oxalate content in whole grain than in refined grain cereals suggests that oxalic acid is primarily located in the outer layers of cereal grains. Cereals and cereal products contribute to the daily oxalate intake to a considerable extent. Vegetarian diets may contain high amounts of oxalate when whole grain wheat and wheat products are ingested. Recommendations for prevention of recurrence of calcium oxalate stone disease have to take into account the oxalate content of these foodstuffs.     

Development of a new method for the complete extraction of carotenoids from cereals with special reference to durum wheat (Triticum durum Desf.)

Due to the growing interest in the role of carotenoids in human health, their qualitative and quantitative analysis in foods is becoming more and more important. High-performance liquid chromatography has become the method of choice for the determination of these phytochemicals. A crucial step prior to the chromatographic separation is the quantitative extraction from the food matrix which was proven to be impeded in durum wheat. To optimize the extraction procedure, several factors with influence on extractability of carotenoids were investigated. Finally, it was shown that soaking of samples in water for 5 min prior to extraction with organic solvents had the strongest impact on extraction yield and led to the most rapid and gentle method. Contents of carotenoids in the extracts of several durum wheat and corn samples were doubled by soaking in water before extracting with methanol/tetrahydrofuran (1/1, v/v). In light of these findings, literature data on contents of carotenoids in cereal grains have to be viewed critically regarding the extraction procedures employed.     

Evaluation of some carotenoids in grapes by reversed- and normal-phase liquid chromatography: a qualitative analysis

Carotenoids in grapes of three Port winemaking cultivars were investigated. Extracts were obtained with n-hexane/diethyl ether mixtures (0/100; 20/80; 50/50; 100/0) and analyzed by normal and reversed phase HPLC-DAD. Selection and identification of peaks were based on spectroscopic characteristics - lambda(max), (%III/II) and k' values, leading to 28 probable carotenoids. Using pure standards, it was possible to identify seven compounds previously described (neochrome, neoxanthin, violaxanthin, flavoxanthin, zeaxanthin, lutein, and beta-carotene), one more type of neochrome reported here, for the first time, and in addition, two geometrical isomers of lutein and beta-carotene were tentatively described. The remaining 17 need to be further identified. High polarity solvent mixtures lead to qualitatively richer chromatograms. Reversed-phase separations allowed the detection of flavoxanthin and the possible geometrical isomer(s) of beta-carotene. Under normal phase, zeaxanthin was detected, and neochromes were better separated from neoxanthin. Extraction with 50/50 n-hexane/diethyl ether mixtures and reversed-phase conditions was the best combination for analysis of the carotenoids, known as precursors of compounds with high aroma impact in wines.     

Antioxidant profiling of native Andean potato tubers (Solanum tuberosum L.) reveals cultivars with high levels of beta-carotene, alpha-tocopherol, chlorogenic acid, and petanin

The antioxidant profile of 23 native Andean potato cultivars has been investigated from a human nutrition perspective. The main carotenoid and tocopherol compounds were studied using high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) and a fluorescence detector, respectively, whereas polyphenols (including anthocyanins in colored tubers) were identified by means of both HPLC-mass spectrometry and HPLC-DAD. Antioxidant profiling revealed significant genotypic variations as well as cultivars of particular interest from a nutritional point of view. Concentrations of the health-promoting carotenoids, lutein and zeaxanthin, ranged from 1.12 to 17.69 microg g(-1) of dry weight (DW) and from 0 to 17.7 microg g(-1) of DW, with cultivars 704353 and 702472 showing the highest levels in lutein and zeaxanthin, respectively. Whereas beta-carotene is rarely reported in potato tubers, remarkable levels of this dietary provitamin A carotenoid were detected in 16 native varieties, ranging from 0.42 to 2.19 microg g(-1) of DW. The amounts of alpha-tocopherol found in Andean potato tubers, extending from 2.73 to 20.80 microg g(-1) of DW, were clearly above the quantities generally reported for commercial varieties. Chlorogenic acid and its isomers dominated the polyphenolic profile of each cultivar. Dark purple-fleshed tubers from the cultivar 704429 contained exceptionally high levels of total anthocyanins (16.33 mg g(-1) of DW). The main anthocyanin was identified as petanin (petunidin-3-p-coumaroyl-rutinoside-5-glucoside). The results suggest that Andean potato cultivars should be exploited in screening and breeding programs for the development of potato varieties with enhanced health and nutritional benefits.