Purification and characterization of a corrinoid compound from Chlorella tablets as an algal health food

Vitamin B(12) content of an algal health food, Chlorella tablets (Chlorella sp.), was determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor-chemiluminescence methods. The values of 200.9-211.6 microg/100 g dry weight determined by the chemiluminescence method were similar to the values (201.3-285.7 microg/100 g dry weight) determined by the microbiological method. A corrinoid compound was purified to homogeneity from the Chlorella tablets and characterized. The purified corrinoid compound was identified as vitamin B12, on the basis of silica gel 60 TLC, C18 reversed-phase HPLC, 1H NMR spectroscopy, and UV-Vis spectroscopy.     

Pseudovitamin B(12) is the predominant cobamide of an algal health food, spirulina tablets.

The vitamin B(12) concentration of an algal health food, spirulina (Spirulina sp.) tablets, was determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor chemiluminescence methods. The values determined with the microbiological method were approximately 6-9-fold greater in the spirulina tablets than the values determined with the chemiluminescence method. Although most of the vitamin B(12) determined with the microbiological method was derived from various vitamin B(12) substitutive compounds and/or inactive vitamin B(12) analogues, the spirulina contained a small amount of vitamin B(12) active in the binding of the intrinsic factor. Two intrinsic factor active vitamin B(12) analogues (major and minor) were purified from the spirulina tablets and partially characterized. The major (83%) and minor (17%) analogues were identified as pseudovitamin B(12) and vitamin B(12), respectively, as judged from data of TLC, reversed-phase HPLC, (1)H NMR spectroscopy, ultraviolet-visible spectroscopy, and biological activity using L. leichmannii as a test organism and the binding of vitamin B(12) to the intrinsic factor.     

Dried green and purple lavers (Nori) contain substantial amounts of biologically active vitamin B(12) but less of dietary iodine relative to other edible seaweeds

Vitamin B(12) concentrations of dried green (Enteromorpha sp.) and purple (Porphyra sp.) lavers (nori) were determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor chemiluminescence methods. The values determined by using the microbiological method (63.58 +/- 2.90 and 32.26 +/- 1.61 microg/100 g of dry weight) were identical to those found by using the chemiluminescence method (69.20 +/- 2.21 and 25.07 +/- 0.54 microg/100 g of dry weight) in both dried green and purple lavers, respectively. A silica gel 60 thin-layer chromatography of both laver extracts shows that non-coenzyme forms (hydroxo and cyano forms) of vitamin B(12) predominate in both dried lavers. The dried lavers contained lesser amounts of dietary iodine ( approximately 4-6 mg/100 g of dry weight) relative to other seaweeds, suggesting that excessive intake of the dried lavers is unlikely to result in harmful intake of dietary iodine. These results indicate that the dried lavers (nori) are the most excellent source of vitamin B(12) among edible seaweeds, especially for strict vegetarians.     

Inactive corrinoid-compound significantly decreases in Spirulina platensis grown in a cobalt-deficient medium.

Spirulina platensis NIES-39 was grown under open culture system in the presence or absence of CoSO(4) (12 microg/L) and/or vitamin B(12) (10 microg/L) to confirm whether CoSO(4) and/or vitamin B(12) stimulate or are essential for growth of the algal cells and for accumulation of vitamin B(12). The addition of CoSO(4) and/or vitamin B(12) could not affect both cell growth and cell yield of the alga. The amount of corrinoid-compound was increased significantly by the addition of CoSO(4) but not by vitamin B(12). A C18 reversed-phase HPLC pattern of the Spirulina corrinoid-compound increased by the addition of CoSO(4) was identical to that of authentic pseudovitamin B(12), which is inactive for human. These results indicate that the algal cells grown in the absence of CoSO(4) are suitable for use of human health foods because the inactive corrinoid-compound can be reduced significantly.     

Characterization of a vitamin B12 compound from unicellular coccolithophorid alga (Pleurochrysis carterae).

A unicellular coccolithophorid alga, Pleurochrysis carterae, contained 125.4 +/- 1.2 microg of vitamin B12 per 100 g dry cell weight of the lyophilized algal cells. A vitamin B12 compound was purified from the lyophilized algal cells and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified pink-colored compound were identical to those of authentic vitamin B12, but not those of vitamin B12 analogues inactive for humans. When 22-week-old B12-deficient rats which excreted substantial amounts of methylmalonic acid (75.5 +/- 12.3 mg/day) in urine were fed the P. carterae (10 g per kg diet)-supplemented diet for 12 d, urinary methylmalonic acid excretion (as an index of vitamin B12 deficiency) of the rats became undetectable and hepatic vitamin B12 level of the rats was significantly increased.     

Characterization of corrinoid compounds from a Japanese black tea (Batabata-cha) fermented by bacteria

A Japanese fermented black tea (Batabata-cha) contained a considerable amount of vitamin B(12) (456 +/- 39 ng per 100 g dry tea leaves and 2.0 +/- 0.3 ng per 100 mL of tea drink). A corrinoid compound was partially purified and characterized from the tea leaves. The patterns of the purified compound by the silica gel 60 thin-layer chromatography and C18 reversed phased high-performance liquid chromatography were identical to those of authentic vitamin B(12). When 20 week old vitamin B(12) deficient rats, which excreted substantial amounts (about 250 mg/day) of methylmalonic acid in urine as an index of vitamin B(12) deficiency, were fed the tea drink (50 mL/day, 1 ng of vitamin B(12)) for 6 weeks, urinary methylmalonic acid excretion (169 +/- 29 mg/day) of the tea drink-supplemented 26 week old rats decreased significantly relative to that (250 +/- 32 mg/day) of the deficient rats. The results indicate that the vitamin B(12) found in the fermented black tea is bioavailable in mammals.     

Certification of B-group vitamins (B1, B2, B6, and B12) in four food reference materials

In 1989, the Community Bureau of Reference started a research program to improve the quality of vitamin analysis in food. To achieve this task, vitamin methodology was evaluated and tested by interlaboratory studies and the preparation of certified reference materials, which will be used for quality control of vitamin measurements. The main improvements in methodology were achieved by testing and standardizing the extraction condition and enzymatic hydrolysis procedures. Results for each individual material are derived from five replicate determinations using at least two independent methods: liquid chromatography (HPLC) and microbiological assay for vitamins B1, B2, and B6; and radioprotein binding and microbiological assays for vitamin B12. The certificate of analysis for four reference materials gives mass fraction values for water-soluble vitamins. These certified values were based on the acceptable statistical agreement of results from collaborating laboratories. Certified values with uncertainties (mg/kg dry matter) for each CRM are as follows: 4.63 (0.20) and 4.10 (0.51) for vitamins B1 and B6, respectively, in CRM 121 (wholemeal flour); 6.51 (0.24), 14.54 (0.3), 6.66 (0.43), and 0.034 (0.003) for vitamins B1, B2, B6, and B12, respectively, in CRM 421 (milk powder); 3.07 (0.17) and 4.80 (0.40) for vitamins B1 and B6, respectively, in CRM 485 (lyophilized mixed vegetables), and 8.58 (0.55), 106.8 (2.8), 19.3 1.5), and 1.12 (0.044) for vitamins B1. B2, B6, and B12, respectively, in CRM 487 (lyophilized pig liver).     

Determination of vitamin B12 in dry feeds by atomic absorption spectrophotometry.

Vitamin B12 was determined in dry feeds by atomic absorption spectrophotometry (AAS). Samples containing B12 were extracted with an assay solution, 5 g EDTA was added to the filtrate, the pH was adjusted to 7 with NH4OH, and 5 g charcoal was added. The charcoal was removed by filtering through ashless paper which was then placed in a beaker and ashed at 600 degrees C. After dissolving the cobalt oxide from the ash in 5N HNO3, cobalt content was determined by using AAS. To determine mg B12/lb feed, ppm cobalt in the feed is multiplied by 10.43. The sensitivity of the proposed procedure is 1 mg vitamin B12/lb. The procedure is rapid and precise, and results compare favorably with AOAC method 43.109.     

Broth from canned clams is suitable for use as an excellent source of free vitamin B12

Vitamin B(12) was assayed and characterized in the broth of canned clams (boiled plain). The broth contained considerable amounts of vitamin B(12) (2.7-14.1 μg/100 g, 1.3-6.7 μg/can). HPLC and LC/ESI-MS/MS chromatograms demonstrated that the clam broth contained true vitamin B(12). Gel filtration experiments indicated that most (72%) of the vitamin B(12) found in the broth was recovered in free vitamin B(12) fractions. These results indicate that the clam broth would be suitable for use an excellent source of free vitamin B(12) for elderly persons with food-bound vitamin B(12) malabsorption.     

Determination of spinosad and its metabolites in citrus crops and orange processed commodities by HPLC with UV detection

Spinosad is an insect control agent that is derived from a naturally occurring organism and is effective on a wide variety of crops, including citrus crops. A method is described for the determination of spinosad and its metabolites in citrus crops and orange processed commodities. The method determines residues of the active ingredients (spinosyns A and D) and three minor metabolites (spinosyn B, spinosyn K, and N-demethylspinosyn D). For dried orange pulp and orange oil, the method has a limit of quantitation (LOQ) of 0.02 microg/g and a limit of detection (LOD) of 0.006 microg/g. For all other sample matrices (whole fruit, edible fruit, juice, and peel), the method has an LOQ of 0.01 microg/g and an LOD of 0.003 microg/g. The analytes are extracted from the various sample types using appropriate solvents, and the extracts are purified by liquid-liquid partitioning and/or solid-phase extraction. All five analytes are determined simultaneously in the purified extracts by reversed-phase high-performance liquid chromatography with ultraviolet detection at 250 nm.     

Major phenolics in apple and their contribution to the total antioxidant capacity

The contribution of each phytochemical to the total antioxidant capacity of apples was determined. Major phenolic phytochemicals of six apple cultivars were identified and quantified, and their contributions to total antioxidant activity of apples were determined using a 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay and expressed as vitamin C equivalent antioxidant capacity (VCEAC). Average concentrations of major phenolics and vitamin C in six apple cultivars were as follows (mg/100 g of fresh weight of apples): quercetin glycosides, 13.20; procyanidin B(2), 9.35; chlorogenic acid, 9.02; epicatechin, 8.65; phloretin glycosides, 5.59; vitamin C, 12.80. A highly linear relationship (r (2) > 0.97) was attained between concentrations and total antioxidant capacity of phenolics and vitamin C. Relative VCEAC values of these compounds were in the order quercetin (3.06) > epicatechin (2.67) > procyanidin B(2) (2.36) > phloretin (1.63) > vitamin C (1.00) > chlorogenic acid (0.97). Therefore, the estimated contribution of major phenolics and vitamin C to the total antioxidant capacity of 100 g of fresh apples is as follows: quercetin (40.39 VCEAC) > epicatechin (23.10) > procyanidin B(2) (22.07) > vitamin C (12.80) > phloretin (9.11) > chlorogenic acid (8.75). These results indicate that flavonoids such as quercetin, epicatechin, and procyanidin B(2) rather than vitamin C contribute significantly to the total antioxidant activity of apples.     

Issues in assessing the validity of nutrient data obtained from a food-frequency questionnaire: folate and vitamin B12 examples

OBJECTIVE: To compare methods used to assess the validity of nutrient intake data obtained from a food-frequency questionnaire (FFQ), using folate and vitamin B12 as nutrient examples. DESIGN: Cross-sectional sample from a population cohort. SETTING: Two postcode areas west of Sydney, Australia. SUBJECTS: In total, 2895 people aged 49 years and older provided dietary data using a semi-quantitative FFQ (79% of 3654 subjects examined). The validity of the FFQ was assessed against three 4-day weighed food records (WFRs) completed by 78 people (mean age 70 years). RESULTS: Folate and vitamin B12 validity data were assessed using different methods. The Spearman ranked correlations (energy-adjusted) were 0.66 for folate and 0.38 for vitamin B12. Using the Bland-Altman method, following loge transformation, no linear trend existed between the differences and means for folate and vitamin B12. Large differences existed between the FFQ and WFR in individual cases, particularly for vitamin B12. Finally, data were divided into quintile categories for the test and reference method: 79% classified folate within one quintile, 65% classified vitamin B12 within one quintile; there was no gross misclassification for folate and only 3% misclassification for vitamin B12. CONCLUSIONS: Different methods of analysis provided different information about the validity of the FFQ. Correlation coefficients should not be used alone to assess the validity of nutrient data, but should be used in conjunction with Bland-Altman analyses. Depending on the use of the data, additional assessment of classification categories is recommended. This worked example demonstrates that absolute intakes of folate and vitamin B12 should be used with caution.     

Contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms

The aim of the study was to determine the contents of mineral elements (Ca, K, Mg, Na, P, Cu, Fe, Mn, Cd, Pb, and Se), vitamins (B(1), B(2), B(12), C, D, folates, and niacin), and certain phenolic compounds (flavonoids, lignans, and phenolic acids) in the cultivated mushrooms Agaricus bisporus/white, Agaricus bisporus/brown, Lentinus edodes, and Pleurotus ostreatus. Selenium, toxic heavy metals (Cd, Pb), and other mineral elements were analyzed by ETAAS, ICP-MS, and ICP methods, respectively; vitamins were detected by microbiological methods (folates, niacin, and vitamin B(12)) or HPLC methods (other vitamins), and phenolic compounds were analyzed by HPLC (flavonoids) or GC--MS methods (lignans and phenolic acids). Cultivated mushrooms were found to be good sources of vitamin B(2), niacin, and folates, with contents varying in the ranges 1.8--5.1, 31--65, and 0.30--0.64 mg/100 g dry weight (dw), respectively. Compared with vegetables, mushrooms proved to be a good source of many mineral elements, e.g., the contents of K, P, Zn, and Cu varied in the ranges 26.7--47.3 g/kg, 8.7--13.9 g/kg, 47--92 mg/kg, and 5.2--35 mg/kg dw, respectively. A. bisporus/brown contained large amounts of Se (3.2 mg/kg dw) and the levels of Cd were quite high in L. edodes (1.2 mg/kg dw). No flavonoids or lignans were found in the mushrooms analyzed. In addition, the phenolic acid contents were very low.     

Apple peels as a value-added food ingredient

There is some evidence that chronic diseases, such as cancer and cardiovascular disease, may occur as a result of oxidative stress. Apple peels have high concentrations of phenolic compounds and may assist in the prevention of chronic diseases. Millions of pounds of waste apple peels are generated in the production of applesauce and canned apples in New York State each year. We proposed that a valuable food ingredient could be made using the peels of these apples if they could be dried and ground to a powder without large losses of phytochemicals. Rome Beauty apple peels were treated with citric acid dips, ascorbic acid dips, and blanches before being oven-dried at 60 degrees C. Only blanching treatments greatly preserved the phenolic compounds, and peels blanched for 10 s had the highest total phenolic content. Rome Beauty apple peels were then blanched for 10 s and dried under various conditions (oven-dried at 40, 60, or 80 degrees C, air-dried, or freeze-dried). The air-dried and freeze-dried apple peels had the highest total phenolic, flavonoid, and anthocyanin contents. On a fresh weight basis, the total phenolic and flavonoid contents of these samples were similar to those of the fresh apple peels. Freeze-dried peels had a lower water activity than air-dried peels on a fresh weight basis. The optimal processing conditions for the ingredient were blanching for 10s and freeze-drying. The process was scaled up, and the apple peel powder ingredient was characterized. The total phenolic content was 3342 +/- 12 mg gallic acid equivalents/100 g dried peels, the flavonoid content was 2299 +/- 52 mg catechin equivalents/100 g dried peels, and the anthocyanin content was 169.7 +/- 1.6 mg cyanidin 3-glucoside equivalents/100 g dried peels. These phytochemical contents were a significantly higher than those of the fresh apple peels if calculated on a fresh weight basis (p < 0.05). The apple peel powder had a total antioxidant activity of 1251 +/- 56 micromol vitamin C equivalents/g, similar to fresh Rome Beauty peels on a fresh weight basis (p > 0.05). One gram of powder had an antioxidant activity equivalent to 220 mg of vitamin C. The freeze-dried apple peels also had a strong antiproliferative effect on HepG(2) liver cancer cells with a median effective dose (EC(50)) of 1.88 +/- 0.01 mg/mL. This was lower than the EC(50) exhibited by the fresh apple peels (p < 0.05). Apple peel powder may be used in a various food products to add phytochemicals and promote good health.     

Quantitative determination of beta-carotene stereoisomers in fresh, dried, and solar-dried mangoes (Mangifera indica L.)

A rapid method for quantitative determination of beta-carotene, including cis-isomers, in dried mango has been developed. Applicability of available methods to dried products was limited because of formation of artifacts caused by extraction and preparation. The analytical procedure was based on the extraction of carotenoids from dried mango mesocarp using a mixture of methanol and acetone/hexane, allowing the separation of disturbing fibers. No saponification was required. Furthermore, carotenoid determination by HPLC on a C30 stationary phase was achieved. This method was applied to determine beta-carotene and its stereoisomers in fresh, dried, and solar-dried mango slices of four cultivars. Drying resulted in a complete and partial degradation of xanthophylls and all-trans-beta-carotene, respectively. Isomerization was shown to depend on the drying process. Whereas conventionally dried mangoes were characterized by elevated amounts of 13-cis-beta-carotene, solar-dried mango slices contained additional amounts of the 9-cis-isomer. Calculation of vitamin A values was based on the real amount of the beta-carotene stereoisomers and ranged from 113 to 420 and from 425 to 1010 RE/100 g for fresh and dried mango slices, respectively.     

Enzymatic determination of free glutamic acid in dried soups and in minced sausages: NMKL collaborative study.

An enzymatic method for the determination of free glutamic acid in meat products and dried soups was collaboratively studied in 11 laboratories. In the presence of the enzyme glutamate dehydrogenase, L-glutamic acid is oxidatively deaminated by nicotinamide adenine dinucleotide (NAD) to 2-oxoglutarate. In a reaction catalyzed by diaphorase, the NADH thus formed converts 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride to a formazan, which is measured in the visible range at 492 nm. Fourteen samples (7 samples of minced sausage and 7 samples of dried cauliflower soup) with glutamate contents varying between 0.4 and 16 g/kg were included in the study. Materials were distributed to participants as blind duplicates and as split level pairs. The mean relative standard deviation (RSDR) for reproducibility for the dried soup material containing glutamate between 7 and 16 g/kg was 4.6%. RSDR values for samples of minced sausage containing glutamate at lower levels (0.4-1.3 g/kg) were between 12 and 16%.     

Direct fluorometric determination of fluorescent substances in powders: the case of riboflavin in cereal flours

Front-face emission spectra of powders can be recorded with a commercial spectrofluorometer. By combining the emissions of a scatterer powder and of a wheat flour sample, the scattering contribution to the front-face emission spectra of flour is removed, and the fluorescence of the flour is isolated. The fluorescence depends on the concentration of the fluorophores. By choosing convenient measurement parameters and by measuring the emission spectra of flour samples suitably enriched with riboflavin, the fluorescence of riboflavin could be isolated from that of other substances present in flours and the concentration of vitamin B(2) in native substrates could be determined. This method is particularly apt for the measurement of vitamin B(2) in low riboflavin-containing powders such as wheat flours, which are usually analyzed through complex chemical and microbiological methods. The method is essentially phenomenological, in view of the interpretation difficulties connected to the origin of the fluorescence resulting from the absorption of multiply scattered photons.     

Difference in folate content of green and red sweet peppers (Capsicum annuum) determined by liquid chromatography-mass spectrometry

Folic acid (pteroylmonoglutamic acid) is used in enriched foods; however, very little folic acid occurs naturally in fruits and vegetables. For the U.S. Department of Agriculture's National Food and Nutrient Analysis Program, a number of fruits and vegetables have been assayed for endogenous folates, by a liquid chromatography-mass spectrometry method, to evaluate the accuracy of existing data for total folate determined by standard microbiological analysis. Folate in red and green sweet peppers (Capsicum annuum) differed notably (70.2 and 20.7 microg/100 g, respectively) and exceeded existing values determined by microbiological assay (18 and 11 microg/100 g, respectively). 5-Methyltetrahydrofolate was the predominant vitamer, but a significant amount of 5-formyltetrahydrolfolate and some 10-formylfolate were present. These findings may assist in making dietary recommendations or developing research diets related to folate. The data from this study have been used to update the folate values in release 19 of the USDA Nutrient Database for Standard Reference.     

Genotype and environment effects on the contents of vitamins B1, B2, B3, and B6 in wheat grain

The total contents of thiamine (vitamin B1), riboflavin (B2), and pyridoxine (B6) and the bioavailable forms of niacin (B3) were determined on wholemeal flours of 24 winter wheat varieties grown on four sites (United Kingdom, Poland, France, and Hungary) in 2007 and of two spring varieties grown on the same sites with the exception of Poland. The contents of vitamins B1 (5.53-13.55 μg/g dw), B2 (0.77-1.40 μg/g dw), and B6 (1.27-2.97 μg/g dw) were within the ranges reported previously, while the content of bioavailable vitamin B3 (0.16-1.74 μg/g dw) was about 10-15% of the total contents of vitamin B3 reported in previous studies. Strong correlations were observed between the contents of vitamins B1, B3, and B6, and partitioning of the variance in the contents of these three B vitamins showed that between 48 and 70% was accounted for by the environment. By contrast, the content of vitamin B2 was not correlated with the contents of other B vitamins, and 73% of the variance was ascribed to the error term, which suggests that this trait may be influenced by genotype × environment interactions. Whereas the contents of vitamins B1, B3, and B6 were correlated positively with the mean temperature from heading to harvest (r > 0.8), the content of vitamin B2 was positively correlated with precipitation during the 3 months prior to heading. These results are discussed in relation to the development of new wheat varieties with enhanced health benefits.     

不同储藏条件对干辣椒品质的影响

为给辣椒干经营者和消费者提供干辣椒在储藏期间营养品质的变化趋势及具体量值,使其了解影响干辣椒储藏的主要因素,从而对干辣椒进行科学的储藏,减少因储藏带来的各种损失。该研究以线辣椒品种（系）“L14”和“陕椒2006”为试材,在冷藏遮光、常温遮光、常温不遮光条件下储藏8个月,定期观测其主要品质性状的变化。结果表明：随着储藏时间的延长,“L14”和“陕椒2006”果实中的辣椒素、辣椒红色素、维生素C和可溶性蛋白质的含量均下降,可溶性糖含量则缓慢上升。用邓肯氏新复极差法对试验数据进行差异显著性检验,发现温度对储藏期间辣椒素、辣椒红色素的影响在储藏150?d后达到显著水平（P＜0.05）,对维生素C及可溶性糖的影响分别在储藏30?d后达到显著（P＜0.05）和极显著水平（P＜0.01）,对其他指标的影响不显著;光对辣椒红色素、维生素C的影响在储藏30?d后达到极显著水平（P＜0.01）,对其他指标的影响不显著。综合各品质指标考虑,低温遮光是辣椒干储藏的理想方式;但从经济的角度考虑,150?d以内的短期储藏在常温遮光条件下进行亦可。