Digestive stability, micellarization, and uptake by Caco-2 human intestinal cell of chlorophyll derivatives from different preparations of pea (Pisum sativum L.)

The digestive stability, efficiency of micellarization, and cellular accumulation of the chlorophyll pigments of different preparations of pea were investigated, using an in vitro digestion procedure coupled with human intestinal Caco-2 cells. Fresh pea (FP), cooked fresh pea (CFP), frozen pea (FZP), cooked frozen pea (CFZP), and canned pea (CP) were subjected to simulated digestion. Although after digestion the pigment profile was modified for all samples, except CP, allomerization reactions and greater destruction of chlorophylls were observed in only FP, which should be due to enzymes in FP that were denaturalized in the rest of the test foods. A pigment extract of CFZP was also subjected to in vitro digestion, showing a positive effect of the food matrix on the pigment digestive stability. The transfer of the chlorophyll pigments from the digesta to the micellar fraction was significantly more efficient in CFZP (57%, p < 0.0001), not significantly ( p > 0.05) different between CFP, FZP, and CP (28-35%), and lowest in FP (20%). Pheophorbide a stood out as the most-micellarized chlorophyll derivative in all of the samples, reaching levels of up to 98%. Incubation of Caco-2 cells with micellar fractions at the same concentration prepared from each test food showed that pigment absorption was considerably lower ( p < 0.006) in cells incubated with FP, whereas there were no differences among the rest of the preparations. Therefore, factors associated with the food matrix could inhibit or mediate the chlorophyll pigment absorption. These results demonstrated that the industrial preservation processes of peafreezing and canningas well as the cooking have a positive effect on the bioaccessibility and bioavailability of the chlorophyll pigments with respect to the FP sample, emphasizing CFZP with greater bioaccesibilty degree.     

Identification of betalains from yellow beet (Beta vulgaris L.) and cactus pear [Opuntia ficus-indica (L.) Mill.] by high-performance liquid chromatography-electrospray ionization mass spectrometry

Betaxanthins, the yellow-orange water-soluble pigments from yellow beet (Beta vulgaris ssp. vulgaris cv. Bejo Zaden) and cactus pear (Opuntia ficus-indica cv. Gialla) have been investigated using an HPLC system compatible with mass spectrometry. Five novel betaxanthins were found and characterized as the immonium adducts of betalamic acid with serine, gamma-aminobutyric acid, valine, isoleucine, and phenylalanine. To enable concentration of betalain samples, desalting was performed by solid-phase extraction. With this technique, betacyanins could be separated from the betaxanthins using the pH-dependent retention characteristics of red and yellow betalains. The betaxanthin fraction was taken for the preparation of betalamic acid as a precursor for semisynthetic standards. The HPLC method was applied to yellow beet and cactus pear, revealing a more complex betalain profile than described earlier, thus proving its suitability for screening of betaxanthin-containing plants as potential sources for natural food colors.     

Effect of microfluidization on in vitro micellization and intestinal cell uptake of lutein from Chlorella vulgaris

Chlorella is a nutrient-rich microalga that contains protein, lipid, minerals, vitamins, and high levels of lutein. This study evaluated the bioavailability of lutein from Chlorella vulgaris using a coupled in vitro digestion and human intestinal Caco-2 cell model. Lutein bioaccessibility was low, and approximately 75% of total C. vulgaris lutein was not micellized during the digestion process but remained in the insoluble digestate. Microfluidization improved lutein micellization efficiency during C. vulgaris digestion. C. vulgaris was microfluidized at a pressure exceeding 10000 psi, and the cell surface disruption was visualized by scanning electron microscopy. The mean C. vulgaris particle size was reduced from 3.56 to 0.35 μm with the microfluidization treatment. C. vulgaris microfluidization at 20000 psi was three times more efficient for aqueous lutein micelles production as compared with untreated C. vulgaris, and the final lutein content accumulated by intestinal Caco-2 cells was also higher with microfluidization. C. vulgaris lutein stability was not affected by microfluidization. These results indicate that microfluidization may be useful for improving lutein bioaccessibility from C. vulgaris during food processing.     

Stability and bioaccessibility of isoflavones from soy bread during in vitro digestion

The impact of simulated digestion on the stability and bioaccessibility of isoflavonoids from soy bread was examined using simulated oral, gastric, and small intestinal digestion. The aqueous (bioaccessible) fraction was isolated from digesta by centrifugation, and samples were analyzed by high-performance liquid chromatography (HPLC). Isoflavonoids were stable during simulated digestion. Partitioning of aglycones, acetylgenistin, and malonylgenistin into the aqueous fraction was significantly (P < 0.01) affected by the concentration of bile present during small intestinal digestion. Omission of bile resulted in nondetectable genistein and <40% of total daidzein, glycitein, and acetylgenistin in the aqueous fraction of digesta. Partitioning of these compounds into the aqueous fraction was increased by physiological concentrations of bile extract. These results suggest that micellarization is required for optimal bioaccessibility of isoflavonoid aglycones. We propose that the bioavailability of isoflavones from foods containing fat and protein may exceed that of supplements due to enhanced bile secretion.     

Identification and quantification of betalains from the fruits of 10 mexican prickly pear cultivars by high-performance liquid chromatography and electrospray ionization mass spectrometry

Qualitative and quantitative analyses of betalain pigments in 10 cultivars/lines of prickly pear (Opuntia spp.) fruit grown in Mexico were conducted with reverse phase high-performance liquid chromatography-diode array detection (HPLC-DAD) coupled with electrospray mass spectrometry (ESI-MS). Betacyanins and betaxanthins were identified by comparison with the UV/vis and mass spectrometric characteristics as well as the retention times of semisynthesized reference betaxanthins. Data revealed that the ratio and concentration of betalain pigments are responsible for the color in the different cultivars, showing the highest betalains content in the fruit of purple colored Camuesa (O. robusta Wendl.) (8.1 mg/g dry fruit), which is comparable to that found in red beet Beta vulgaris L. ssp. Var. Pablo) (8.6 mg/g dry tissue). Yellow betalains were absent in Reyna (O. alba-carpa) prickly pear cultivar. A total of 24 known/unknown betalains were present in the prickly pear fruit samples studied, including 18 betaxanthins and 6 betacyanins. Our results indicate that prickly pear cultivars can be considered as a potential source of yellow and red natural colorants.     

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.     

Betalains in red and yellow varieties of the Andean tuber crop ulluco (Ullucus tuberosus)

The betalain pigments in ulluco (Ullucus tuberosus), a tuberous crop native to the Andes, have been investigated for the first time using LC-DAD-ESI-MS-MS(2) analyses. Five red, yellow, and red-spotted accessions introduced into New Zealand as a new food crop plus two red tetraploid lines were investigated. Thirty-two different betalains were identified. Both the yellow and red tubers were rich in yellow betaxanthins, and the most prominent among the 20 identified were histidine-betaxanthin, arginine-betaxanthin and glutamine-betaxanthin. Arginine-betaxanthin has been reported to occur naturally only once before and was found in yellow ulluco but not in the red tubers. Twelve betacyanins were found in red tubers, with roughly 50% of this content being betanin/isobetanin. Betacyanin levels were up to 70 microg/g fresh weight in red tubers, but were below quantifiable levels in yellow tubers. Betaxanthin levels were up to 50 microg/g fresh weight in yellow tubers. Interference by betacyanins in measuring levels of betaxanthins by visible spectrophotometry is discussed. Low concentrations of betalains were detected in leaves, whereas stems contained total levels similar to the tubers, with dopamine-betaxanthin and betanin being the major pigments. This is the first report describing both the betacyanin and betaxanthin patterns in a plant from the Basellaceae family.     

Bioaccessibility of beta-carotene, lutein, and lycopene from fruits and vegetables

Epidemiological studies have consistently demonstrated that there is an association between carotenoid-rich food intakes with a low incidence in chronic diseases. Nevertheless, there is not an association between the intake of total dietary carotenoids and chronic health incidence in the European population, probably because of different carotenoid food sources and bioavailability. The objective of this study was to evaluate the small and large intestine bioaccessibilities of major dietary carotenoids from fruits and vegetables in a common diet. A bioaccessibility model that includes enzymatic digestion and in vitro colonic fermentation was employed. Lutein presented greater small intestine bioaccessibility (79%) than beta-carotene (27%) or lycopene (40%). With regard to large intestine bioaccessibility, similar amounts of lycopene and beta-carotene were released from the food matrix (57%), whereas small amounts of lutein (17%) were released. These results suggest that 91% of the beta-carotene, lutein, and lycopene contained in fruits and vegetables is available in the gut during the entire digestion process. Colonic fermentation is shown to be important for carotenoid availability in the gut.     

Formation of decarboxylated betacyanins in heated purified betacyanin fractions from red beet root (Beta vulgaris L.) monitored by LC-MS/MS

Mixtures of mono-, bi-, and tridecarboxylated betacyanins together with their corresponding neobetacyanins obtained from Beta vulgaris L. root juice as heating degradation products of betacyanins were identified by high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) and diode-array (LC-DAD) detection. Two monodecarboxy-betacyanin pairs of diastereomers were detected after the decarboxylation in ethanolic and aqueous solutions. Generation of 17-decarboxy-betacyanins and 2-decarboxy-betacyanins was suggested, the latter so far never having been attributed to betacyanin thermal degradation products. Other main products of decarboxylation were 2,17-bidecarboxybetanin, its isoform, and 14,15-dehydrogenated (neobetacyanin) derivatives of all the decarboxylated betacyanins. The results of this research are crucial in determining betacyanin degradation mechanisms in juices or extracts of B. vulgaris L. roots and other products containing these pigments.     

Effect of milk and brewing method on black tea catechin bioaccessibility

The aim of this study was to investigate whether milk reduces the bioaccessibility of tea catechins, which would compromise tea beneficial effects ascribed to polyphenols. Adding milk to black tea has been shown to lead to polyphenol-protein complexes. So far, data on the intestinal stability of polyphenol-protein complexes are scarce. English black tea (0.93 ± 0.06 mol/L total catechins) and Indian black tea (1.83 ± 0.08 mol/L catechins) were prepared with skimmed or full-fat milk and subjected to simulated gastric, small intestinal, and brush border digestion. Adding milk (5.6-40%) to tea results in a decrease of total catechin (TCAT) recovery. However, the bioaccessibilities of TCAT of tea with milk versus tea controls were comparable (p > 0.05). The type of milk did not influence TCAT recovery during all digestive stages (p > 0.05). Polyphenol-protein complexes are degraded during digestion. It is very unlikely that consumption of tea with or without milk will result in differences in catechin plasma concentration.     

果胶对脂类和类胡萝卜素消化利用影响研究进展

果胶已经被证实可以影响脂类的消化,脂溶性的类胡萝卜素在消化阶段需要被脂滴包裹才能进入小肠形成胶束,因此果胶对类胡萝卜素的消化利用也会存在潜在影响。该文综述了近年来果胶对脂类和类胡萝卜素消化利用影响研究进展,主要分为果胶对消化液黏度的影响、对消化酶的影响、与钙离子的相互作用、与胆盐的结合作用以及对脂滴的包裹作用这5个方面。该文为后续分析如何提高果蔬中类胡萝卜素生物利用度提供理论依据。     

In vitro系统评价胃肠液pH及土液比对铅、镉、砷生物可给性的影响

土壤中铅、镉、砷可能直接通过人的口部无意摄入进入人体,危害人体健康,而各研究者评价土壤重金属对人体健康风险所用的实验参数有别,结果缺乏可比性,因此探讨不同评价方法对结果的影响具有重要意义。以污染的棕钙土和红壤为研究对象,应用in vitro方法研究分析胃肠阶段不同土液比、pH以及土壤性质对铅、镉、砷生物可给性的影响。结果表明,重金属的生物可给性与in vitro系统中的pH、土液比、土壤类型以及重金属本身有关。在胃阶段,随着土液比升高,3种重金属生物可给性趋于降低;不同土液比处理下,红壤中铅生物可给性大于棕钙土,而砷则相反,红壤和棕钙土中镉的生物可给性差异不明显。在肠阶段,随着土液比升高,3种重金属生物可给性也趋于降低（除了红壤镉以外）,土液比1∶100的3种重金属生物可给性均显著大于1∶10。不同pH处理下,铅的生物可给性随pH的升高逐渐降低,而pH对镉和砷的生物可给性的影响与土液比有关。因此,肠胃实际吸收的重金属可能与摄入水量、食物成分与组成以及食物摄入引起的肠液pH变化有关。     

The effect of in vitro digestion on steryl ferulates from rice (Oryza sativa L.) and other grains

Polished and cargo rice, wild rice, rice bran, corn bran, and wheat bran were subjected to a static in vitro digestion model, to monitor changes in their steryl ferulate content and composition. Free sterols, possible hydrolysis products of steryl ferulates, were also measured. Additionally, steryl ferulate bioaccessibility was calculated as the percentage of steryl ferulates liberated from the grain matrix into the digestive juice. Steryl ferulate content ranged between 6.1 and 3900 μg/g and decreased by 1-63% due to digestion. A parallel increase in free sterols of more than 70% was observed in all samples. Additionally, bioaccessibility of steryl ferulates was found to be almost negligible. These findings suggest that intestinal enzymes immediately hydrolyze steryl ferulates, which are liberated from the grain matrix, and thus they are practically unavailable for absorption in the small intestine. This further indicates that the hydrolysis products of steryl ferulates could be bioactive in the gut.     

Chemical stability and colorant properties of betaxanthin pigments from Celosia argentea

The chemical stability and colorant properties of three betaxanthins recently identified from Celosia argentea varieties were evaluated. Lyophilized betaxanthin powders from yellow inflorescences of Celosia exhibited bright yellow color and high color purity with strong hygroscopicity. The aqueous solutions containing these betaxanthins were bright yellow in the pH range 2.2-7.0, and they were most stable at pH 5.5. The betaxanthins in a model system (buffer) were susceptible to heat, and found to be as unstable as red betacyanins (betanin and amaranthine) at high temperatures (>40 degrees C), but more stable at 40 degrees C with the exclusion of light and air. The three betaxanthins had slightly higher pigment retention than amaranthine/isoamaranthine in crude extracts at 22 degrees C, as verified by HPLC analysis. Lyophilized betaxanthins had much better storage stability (mean 95.0% pigment retention) than corresponding aqueous solutions (14.8%) at 22 degrees C after 20 weeks. Refrigeration (4 degrees C) significantly increased pigment retention of aqueous betaxanthins to 75.5%.     

Identification of betalains from petioles of differently colored Swiss chard (Beta vulgaris L. ssp. cicla [L.] Alef. Cv. Bright Lights) by high-performance liquid chromatography-electrospray ionization mass spectrometry

The betalain pattern of differently colored Swiss chard (Beta vulgaris L. ssp. cicla [L.] Alef. cv. Bright Lights) was investigated for the first time. Nineteen betaxanthins and nine betacyanins were identified by RP-HPLC and positive ion electrospray mass spectrometry, co-injection experiments with semisynthetic reference compounds, and standards derived from authentic plant material, respectively. Histamine-betaxanthin and alanine-betaxanthin were found to be novel betaxanthins, which to the best of our knowledge have not been reported as natural compounds until now. Furthermore, tyramine-betaxanthin (miraxanthin III) and 3-methoxytyramine-betaxanthin, which to date were known only from families other than the Chenopodiaceae, were detected for the first time in colored Swiss chard. The betacyanin pattern of purple petioles was composed of betanin, isobetanin, betanidin, and isobetanidin. Although phyllocactin was present in only trace amounts, further acylated structures such as betanidin-monoferuloyl-5-O-beta-diglucoside and lampranthin II, accompanied by their corresponding C(15)-epimers, were identified. In addition, quantification of betalains and CIE LCh degrees measurements were performed with the colored extracts to correlate the visual appearance with the respective pigment patterns. Besides the novel phytochemical findings, the present study is useful for the evaluation of betalainic Swiss chard as a potential coloring foodstuff.     

Assessment of degradation and intestinal cell uptake of carotenoids and chlorophyll derivatives from spinach puree using an in vitro digestion and Caco-2 human cell model

Although numerous studies have demonstrated the health benefits of chlorophyll derivatives, information regarding the digestion, absorption, and metabolism of these phytochemicals is quite limited. To better understand the digestion of these pigments, green vegetables including fresh spinach puree (FSP), heat- and acid-treated spinach puree (HASP), and ZnCl(2)-treated spinach puree (ZnSP) were subjected to an in vitro digestion method which simulates both the gastric and small intestinal phases of the process. Native chlorophylls were converted to Mg-free pheophytin derivatives during digestion. Conversely, Zn-pheophytins were completely stable during the digestive process. Transfer of lipophilic chlorophyll derivatives, as well as the carotenoids lutein and beta-carotene, into the aqueous micellar fraction from the food matrix was quantified. Micellarization of total chlorophyll derivatives differed significantly (p < 0.05) for FSP (37.6%), HASP (17.2%), and ZnSP (8.7%). Micellarization of chlorophyll a derivatives was determined to be significantly more efficient than chlorophyll b derivatives in FSP and HASP (p < 0.01), but not in ZnSP (p > 0.05). Intestinal cell uptake of micellarized pigments was investigated using HTB-37 (parent) and clonal TC7 lines of human Caco-2 cells. Medium containing the pigment-enriched fraction generated during digestion was added to the apical surface of fully differentiated monolayers for 4 h. Pigments were then extracted from cells and analyzed by C18 HPLC with photodiode array detection. Both Caco-2 HTB-37 and TC7 clone cells accumulated 20-40% and 5-10% of micellarized carotenoid and chlorophyll derivatives, respectively. These results are the first to demonstrate uptake of chlorophyll derivatives by human intestinal cells and to support the potential importance of chlorophylls as health-promoting phytochemicals.     

Bioaccessibility of carotenoids and vitamin E from their main dietary sources

Vitamin E and carotenoids are fat-soluble microconstituents that may exert beneficial effects in humans, including protection against cancer, cardiovascular diseases, and age-related eye diseases. Their bioavailability is influenced by various factors including food matrix, formulation, and food processing. Since human studies are labor-intensive, time-consuming, and expensive, the in vitro model used in this study is increasingly being used to estimate bioaccessibility of these microconstituents. However, the ability of this model to predict bioavailability in a healthy human population has not yet been verified. The first aim of this study was to validate this model by comparing model-derived bioaccessibility data with (i) human-derived bioaccessibility data and (ii) published mean bioavailability data reported in studies involving healthy humans. The second aim was to use it to measure alpha- and gamma-tocopherol, beta-carotene, lycopene, and lutein bioaccessibility from their main dietary sources. Bioaccessibility as assessed with the in vitro model was well correlated with human-derived bioaccessibility values (r = 0.90, p < 0.05), as well as relative mean bioavailability values reported in healthy human groups (r = 0.98, p < 0.001). The bioaccessibility of carotenoids and vitamin E from the main dietary sources was highly variable, ranging from less than 0.1% (beta-carotene from raw tomato) to almost 100% (alpha-tocopherol from white bread). Bioaccessibility was dependent on (i) microconstituent species (lutein > beta-carotene and alpha-carotene > lycopene and alpha-tocopherol generally > gamma-tocopherol), (ii) food matrix, and (iii) food processing.     

Color properties and stability of betacyanins from Opuntia fruits

The colorant properties of pigments from Opuntia stricta, Opuntia undulata, and Opuntia ficus-indicafruits were studied. The pigments were extracted with different solvents and identified by high-performance liquid chromatography. On the basis of their visible light spectra, the pigments were identified as betalains. In O. undulata and O. ficus-indica fruits, both betacyanins and betaxantins were identified, while in O. stricta fruits only betacyanins (betanin and isobetanin) were detected. O. stricta fruits showed the highest betacyanin content (80 mg/100 g fresh fruit). The thermal stability of the pigment extracts was dependent on the pH, with the maximum stability being at pH 5, as expected for betacyanins. At this value and a storage temperature of 4 degrees C, a deactivation half-life time of more than 1 year, with no added stabilizers, was determined. According to these studies, cactus pears from O. stricta may well be considered as a potential source of natural red colorants.     

Development of an in vitro digestion method to assess carotenoid bioavailability from meals.

The objective of this study was to develop a model for assessing the bioavailability of carotenoids from meals using an in vitro digestion procedure. A meal was prepared using baby food carrots, spinach, and a meat, plus tomato paste. The aqueous fraction was isolated from digesta to determine the quantity of carotenoids transferred from the food to micelles. The micellarization of lutein (25-40%) exceeded (p < 0.01) that of alpha- and beta-carotene (12-18%) and lycopene (<0.5%). Micellarization of carotenoids was not affected by elimination of the gastric phase of the digestive process. The absence of bile extract prevented the transfer of carotenoids from foods to micelles, whereas omission of pancreatin only reduced the micellarization of the carotenes. Differentiated cultures of Caco-2 human intestinal cells accumulated 28-46% of micellarized carotenoids from the medium after 6 h. These results support the usefulness of the in vitro digestion process as a rapid and cost-effective model for screening the bioavailability of carotenoids from meals.     

Identification and distribution of simple and acylated betacyanins in the Amaranthaceae

Red-colored plants in the family Amaranthaceae are recognized as a rich source of diverse and unique betacyanins. The distribution of betacyanins in 37 species of 8 genera in the Amaranthaceae was investigated. A total of 16 kinds of betacyanins were isolated and characterized by HPLC, spectral analyses, and MS. They consisted of 6 simple (nonacylated) betacyanins and 10 acylated betacyanins, including 8 amaranthine-type pigments, 6 gomphrenin-type pigments, and 2 betanin-type pigments. Acylated betacyanins were identified as betanidin 5-O-beta-glucuronosylglucoside or betanidin 6-O-beta-glucoside acylated with ferulic, p-coumaric, or 3-hydroxy-3-methylglutaric acids. Total betacyanin content in the 37 species ranged from 0.08 to 1.36 mg/g of fresh weight. Simple betacyanins (such as amaranthine, which averaged 91.5% of total peak area) were widespread among all species of 8 genera. Acylated betacyanins were distributed among 11 species of 6 genera, with the highest proportion occurring in Iresine herbstii (79.6%) and Gomphrena globosa (68.4%). Some cultivated species contained many more acylated betacyanins than wild species, representing a potential new source of these pigments as natural colorants.