Determination of total vitamin B6 in foods by isocratic HPLC: a comparison with microbiological analysis

This paper describes a rapid and sensitive, high performance liquid chromatography (HPLC) method for analysis of vitamin B₆ in various foods. The method is based on isocratic elution and it provides complete separation of the three major B₆-vitamers: pyridoxine, pyridoxal and pyridoxamine within 12 min. Samples of vegetable origin were extracted with mild acid hydrolysis prior to enzymatic digestion with acid phosphatase and β-glucosidase and by analysis of the two digests separately it was possible to distinguish between free pyridoxine and β-glucosylic forms of pyridoxine. Results for several food samples analysed by this method were compared to the results of a microbiological analytical method using Saccharomyces uvarum. The comparison showed a systematic difference in results obtained with the two methods. Vitamin B₆ data from the HPLC method were approximately 70% higher for animal foodstuffs, 20% higher for fruit and vegetables, but approximately 20% lower for grain products than for the microbiological method. Models explaining these differences are discussed.     

Amino Acid and Vitamin Composition of Raw and Cooked Horse Mackerel

Amino acid, vitamin (A, E, B₁, B₂, B₃ and B₆), and proximate composition were determined in raw and cooked horse mackerel. The changes in amino acid, vitamin, and proximate content were found to be significant for all cooking methods (frying, grilling, and steaming). Cooking did in general significantly increase the contents of essential, semi-essential, and other amino acids compared to raw fish species. Amino acid contents of grilled mackerel were significantly higher (p < 0.05) than those found in fried and steamed mackerel. The A, E, B₂, and B₆ vitamin content of fried horse mackerel was found to be significantly (p < 0.05) higher than the grilled and steamed samples. The B₁ content of steamed and B₃ content of grilled were found higher than the other cooked samples. Moisture, protein, fat, ash, and carbohydrate contents of cooked fish ranged between 56.52% to 61.34%, 20.79% and 23.93%, 13.44% and 19.61%, 1.70% and 2.47%, and 1.02% and 4.36 %, respectively. Fried fish had intermediate fat values, while grilled and steamed fishes had a comparatively low value.     

A novel method for determining vitamin B1 in a wide variety of foodstuffs with or without polyphenols

Certain foodstuffs exhibit matrix interference effects on the vitamin B₁ analysis prescribed in the official methods of the European Union, AOAC International, and Japan. In this study, we demonstrated that one of the problematic polyphenols in green tea or cocoa was tannin. For these matrices, thiamine was found to interact with tannin in the extraction step and was thus removed by filtration as a residue. To resolve the matrix interference, we proposed lowering the pH of the extraction solution by adding hydrochloric acid after the enzyme reaction. High precision (2–3% RSDr) and good recovery (98.3–103%) were obtained with reference materials using the proposed method. We also confirmed the equivalence of the obtained data from the proposed method and the Japanese official method for nutritional labeling. From these results, the method was found to be effective for vitamin B₁ analysis regardless of the presence of interference matrices.     

A Simple Liquid Chromatographic Method for Determination of B6 Vitamers in Raw and Cooked Chicken

Four forms of vitamin B₆ (B₆ vitamers) in various chicken tissues were determined by a liquid chromatographic method. The vitamers were extracted from the tissues with metaphosphoric acid and separated on a C₁₈ reverse phase column followed by fluorescence detection. Recoveries of added vitamins at two concentrations were mostly between 84.5% and 107.9% The most abundant form of B₆ in raw tissues was pyridoxal phosphate (PLP) followed by pyridoxamine phosphate (PMP). Cooked tissues contained more PMP than PLP. The levels of pyridoxal (PL) and pyridoxamine (PM) were relatively low. Data also indicated a net increase of PMP and PM vs a decrease of PLP and PL during cooking.     

Effects of supplementary folic acid and vitamin B12 on hepatic metabolism of dairy cows according to methionine supply

The present experiment was undertaken to study the interactions between dietary supplements of rumen-protected methionine (RPM) and intramuscular injections of folic acid and vitamin B₁₂, given from 3 wk before calving to 16 wk of lactation, on hepatic metabolism of lactating dairy cows. Sixty multiparous Holstein cows were assigned to 10 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet calculated to supply Met as 1.83% of metabolizable protein, whereas the 3 other cows were fed the same diet supplemented with 18 g of RPM calculated to provide Met as 2.23% of metabolizable protein. Within each level of Met, the cows received no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid alone or combined with 10 mg of vitamin B₁₂. Liver biopsies were taken at 2, 4, 8, and 16 wk of lactation. Liver concentrations of folates and vitamin B₁₂ were increased by their respective supplements but this response to vitamin supplements was altered by methionine supply. Concentrations of total lipids and triglycerides increased in livers of cows fed RPM, whereas concentrations of cholesterol ester, cholesterol, diglycerides, phosphatidylethanolamine, and phosphatidylcholine were not affected. Folic acid, alone or combined with vitamin B₁₂, tended to increase the ratio of phosphatidylcholine to phosphatidylethanolamine. Gene expression of 5,10-methylene-tetrahydrofolate reductase, microsomal transfer protein, and phosphatidylethanolamine methyltransferase were higher in liver of cows fed RPM supplements. The relative mRNA abundance of 5,10-methylene-tetrahydrofolate reductase and methylmalonyl-CoA mutase were increased by the combined injections of folic acid and vitamin B₁₂, whereas those of methionine synthase and methionine synthase reductase were not affected by treatments. These results suggest that increasing supply of methyl groups, as preformed labile methyl groups or through methylneogenesis, affected the methylation cycle but had a limited effect on dairy cow performance. The observed effects of the combined supplement of folic acid and vitamin B₁₂ on lactational performance of dairy cows probably result from an improvement of energy metabolism during early lactation.     

Determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts

A method for the determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B₆, biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B₆ in various foodstuffs. There was good agreement between vitamin B₆ concentrations determined after 24 h using the WST-8 colorimetric method and those obtained after 48 h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate.     

Influence of methionine supply on the response of lactational performance of dairy cows to supplementary folic acid and vitamin B12

The present experiment was undertaken to determine if the effects of supplementary folic acid on lactational performance were caused by improved methylneogenesis and if the supply in vitamin B₁₂ could affect this metabolic pathway. In this eventuality, supplementary Met, a major source of preformed methyl groups, should reduce the requirements for these vitamins. Sixty multiparous Holstein cows were assigned to 10 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet estimated to supply Met as 1.83% metabolizable protein and 3 cows were fed the same diet supplemented with 18 g of rumen-protected methionine (RPM) to supply Met as 2.23% of metabolizable protein. Within each level of Met, cows received no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid alone or combined with 10 mg of vitamin B₁₂ from 3 wk before to 16 wk after calving. There was no treatment effect on dry matter intake during pre- and postcalving periods: 13.4 ± 0.4 and 21.8 ± 0.4 kg/d, respectively. Milk production was not affected by RPM supplementation. Folic acid and vitamin B₁₂ given together tended to increase milk production during the 16 wk of lactation. This effect was more pronounced during the first 4 wk of lactation: 37.5, 37.7, and 40.3 ± 0.9 kg/d for cows receiving no vitamin supplement, folic acid alone, or folic acid combined with vitamin B₁₂, respectively. Milk fat yield was not affected by treatments. Lactose, crude protein, and total solid yields were greater, in early lactation, in cows injected with folic acid and vitamin B₁₂ together but this effect diminished as lactation progressed. Intramuscular injections of folic acid alone or combined with vitamin B₁₂ tended to decrease plasma concentrations of homocysteine from 5.51 μM with no vitamin supplement to 4.54 and 4.77 ± 0.37 μM, respectively. Results of the present experiment suggest that the effects of the combined supplement of folic acid and vitamin B₁₂ on lactational performance of dairy cows were not due to an improvement in methyl groups supply, because RPM supplement, a source of preformed methyl groups, did not alter the cow responsiveness to vitamin supplements.     

Vitamin fortified rice grain using spraying and soaking methods

The objective of this work was to fortify three types of rice grains (brown, white milled and parboiled) with B complex vitamins (B₁, B₂, B₃, B₅, B₆, B₁₂), using two different physical treatment methods, (1) soaking of whole dehusked or milled rice kernels in vitamin solutions at 90 °C for 15 min and (2) full spraying of whole kernels with vitamin solution at 35 °C, both followed by oven drying. Various nutrient concentrations were added and the vitamin retention of dried fortified as well as of cooked rice were determined. Fortified rice was evaluated based on vitamin retention, estimated as the percentage of vitamin in rice after fortification treatment versus the total amount of added plus its initially vitamin quantity. It was found that the average retention of all vitamins in dried rice was high and varied from 54.3% to 85.3% for the spraying and from 53.5% to 76.2% for the soaking method respectively. After rice cooking, the retention was maintained in sufficient level (>70%) for all vitamins except for B₁₂. When excess of water was used for cooking, the water-soluble vitamins were leached; however a significant amount of vitamins still remained, varying from 13.5% (B₁₂) to 51.2% (B₁). The good retention of vitamins, even after cooking, achieved applying these relatively simple techniques, may be attributed to the absorption and diffusion of vitamins in the interior of rice kernels in high concentrations. Dried fortified rice samples properties were evaluated.     

Milk Drink Using Whey Butter Cheese (queijo manteiga) and Acerola Juice as a Potential Source of Vitamin C

The production of a beverage from whey butter cheese (WBC) and acerola juice (AJ) presents good commercialization potential, uniting the benefits provided by the former with those of latter, including the ingestion of essential amino acids and increasing the vitamin C content, resulting in a product of differentiated nutritive value. Three beverage formulations were developed: B₁ (50% WBC–50% AJ v/v), B₂ (70% WBC–30% AJ, v/v), and B₃ (30% WBC–70% AJ, v/v). Lower protein and carbohydrate values were found for the formulations with less whey (p < 0.05). All the beverages presented an energy value below those declared for dairy beverages commercialized on the Brazilian market. The beverages B₁ and B₂ were considered food products that were sources of vitamin C. B₂ presented significant variations for all the sensory attributes evaluated (p < 0.05), although the results were similar to those obtained for B₁, suggesting the commercialization potential of these formulations.     

Kinetics of free and glycosylated B6 vitamers,thiamin and riboflavin during germination of pea seeds

The effect of germination on the free and glycosylated B₆ vitamers (pyridoxine, pyridoxal, pyridoxamine and glycosylated pyridoxine), thiamin and riboflavin content in pea seed (Pisum sativum L, var Esla) was investigated. In raw peas, the total vitamin B₆ content (sum of all vitamers expressed as pyridoxine) was 2.183 mg kg⁻¹ DM. Pyridoxine was found to be the predominant B₆ vitamer, while only a little amount of glycosylated pyridoxine, the vitamer with the lowest boiavailability, was observed. Thiamin and riboflavin content in raw peas was found to be 7.614 and 2.110 mg kg ⁻¹ DM, respectively. After 6 days of germination, carried out at 20°C, in the dark and with daily rinsing, a significant increase of the total vitamin B₆ content, mainly due to the increase of glycosylated pyridoxine, was observed. The content of pyridoxal and riboflavin increased in peas 7.5 and 2 times, respectively, whereas pyridoxine, pyridoxamine and thiamin decreased by 83, 24 and 25%, respectively, at the end of the experiment. Results obtained for these vitamins during pea germination were fitted to modelling curves in order to predict vitamin content for different germination periods. © 1999 Society of Chemical Industry     

Effect of frozen storage on the degree of vitamin B6degradation in different foods

Investigation of the effect of freezing and storage at-18° C for 5 months on the stability of total vitamin B₆ in foods of different origin leads to the conclusion that the decrease in vitamin B₆ content ranged from 18.92% to 60.26% and that the loss is significantly greater in food of animal origin (an average of 55.0%). Obviously, biostructure, that is chemical composition, is one of the basic factors affecting the degradation degree of vitamin B₆ in foods preserved by freezing. However, in relation of their biological value and with reference to the declaration allowing deep-frozen vegetables to be stored for 12–24 months, some foods of vegetable origin preserved by freezing exhibit a relatively high loss of biological value in terms of vitamin B₆ as early as after 5 months of storage.     

Effect of Feeding Three Levels of Riboflavin, Niacin and Vitamin B6 to Male Chickens on the Nutrient Composition of Broiler Breast Meat

Sixty day-old male chicks were fed three rations differing in riboflavin (R), vitamin B₆, and niacin (N) content. The three rations were: (1) an adjusted basal ration containing R, B₆ and N 10% above National. Research Council recommendations, (2) a ration containing 75% of the 3 vitamins as in (1), and (3) with 125% of the 3 vitamins as in (1). Birds were grown for 51 days and the breast meat harvested and analyzed for proximate composition, 3 vitamins and 9 minerals. No significant differences in the nutrient content of meat were found attributable to the dietary treatments. Results indicated that broiler meat composition was fairly constant even though birds consumed wide differences in R, B₆ and N.     

Vitamin B12 Activity in Miso and Tempeh

The USP microbiological assay with L. leichmannii, ATCC 7830, was used to determine vitamin B₁₂ activity in light rice miso, dark rice miso, barley miso, tempeh and tempeh burger. Unpasteurized misos were found to have the highest B₁₂ content, averaging 0.21 μg/ 100g. Vitamin B₁₂ activity in miso ranged from a high of 0.25 μg/ 100g in barley miso to a low of 0.15 μg/100g in light rice miso. Pasteurized tempeh contained 0.12 μg vitamin B₁₂ per 100g food. Tempeh burger contained 0.06 to 0.11 μg vitamin B₁₂ per 100g food. The variation in vitamin B₁₂ activity found in these products may be due to different conditions used or produced during fermentation. Collaborative studies are needed and assessment of vitamin B₁₂ pseudoform activity before these foods can be considered a source of vitamin B₁₂.     

Apparent ruminal synthesis of B vitamins in lactating dairy cows fed diets with different forage-to-concentrate ratios

Effects of the forage-to-concentrate ratio on apparent ruminal synthesis of thiamine, riboflavin, niacin, vitamin B₆, folates, and vitamin B₁₂ were evaluated in an experiment using 14 ruminally and duodenally cannulated Holstein cows. The experiment was a crossover design with two 15-d treatment periods and a 14-d preliminary period in which cows were fed a diet intermediate in composition between the treatment diets. Treatments were diets containing low-forage (44.8% forage, 32.8% starch, 24.4% neutral detergent fiber) or high-forage (61.4% forage, 22.5% starch, 30.7% neutral detergent fiber) concentrations. Both diets were formulated with different proportions of the same ingredients. Concentrations of B vitamins were analyzed in feed and duodenal digesta. Apparent ruminal synthesis of each B vitamin was calculated as the duodenal flow minus the intake. The high-forage diet had the highest concentrations of riboflavin, niacin, vitamin B₆, and folates, whereas the low-forage diet had the highest thiamine concentration. Vitamin B₁₂ in the diets was under the level of detection. Consequently, despite a reduction in dry matter intake when the cows were fed the high-forage diet, increasing dietary forage concentration increased or tended to increase intakes of riboflavin, niacin, and vitamin B₆ but reduced thiamine and folate intakes. Increasing dietary forage concentration reduced apparent ruminal degradation of thiamine and apparent ruminal synthesis of riboflavin, niacin, and folates and increased ruminal degradation of vitamin B₆, but had no effect on ruminal synthesis of vitamin B₁₂. As a consequence, increasing the forage-to-concentrate ratio had no effect on the amounts of thiamine, riboflavin, and vitamin B₁₂ reaching the small intestine but decreased the amounts of niacin, vitamin B₆, and folates available for absorption. Apparent ruminal syntheses of riboflavin, niacin, folates, and vitamin B₁₂ were correlated positively with the amount of starch digested in the rumen and duodenal flow of microbial N, whereas these correlations were negative for thiamine. Apparent ruminal syntheses of thiamine and vitamin B₆ were negatively correlated with their respective intakes, whereas folate intake was positively correlated with its synthesis in the rumen.     

Quality attributes of a heart healthy yogurt

The objective was to study the effect of heart healthy nutrients on the physico-chemical, microbiological and sensory characteristics of yogurt. Thiamine (vitamin B₁), riboflavin (vitamin B₂), niacin (vitamin B₃), folic acid (vitamin B₉), manganese and magnesium were added during mix preparation at 0%, 30%, 60% and 90% of their respective recommended dietary allowance (RDA). Fiber (Ceolus Fiber DF-17) was added at a constant rate of 176 g/7.570 kg yogurt mix in all the treatments. Total solids in the control were kept constant with non-fat dry milk. Incorporation of the heart healthy nutrients at the 30%, 60% and 90% RDA significantly decreased syneresis, pH, L*, a* values but significantly increased b* value. Product viscosity was significantly increased by the incorporation of the nutrients at 60% of their respective RDA's. The incorporation of the above heart healthy vitamins and minerals at any of the studied rates in yogurts did not significantly affect flavor, appearance, body and texture and microbial counts of the product. Although there were subtle yet significant changes in instrumental color and viscosity these slight changes could not be detected by sensory evaluation. Yogurts can successfully be manufactured with the above heart healthy nutrients.     

Microbial Biosensor System for Rapid Determination of Vitamin B6

A microbial biosensor system was based on an immobilized microorganism (Succharomyces uvarum 4288 ATCC9080) and Clark-type oxygen electrode consisting of a platinum cathode, lead anode, alkaline electrolyte, and an oxygen permeable Teflon membrane. The determination was based on the respiratory activity of the microorganism in presence of vitamin B₆. Optimum conditions were: concentration of immobilized cells on membrane: OD₆₆₀, = 0.05, temperature: 30°C. When oxygen permeable Teflon membranes (0.5 mil and 2.0 mil) were employed, relationships between vitamin B₆, (pyridoxine) and current decrease were linear in the range of 0.5 ng/mL-2.5 ng/mL and 2.5 ng/mL-12.5 ng/mL, respectively. One assay could be completed within 15 min. This system provided rapid and simple determinations of vitamin B₆ in marine products with good correlation to traditional microbial assay values.     

Effect of Cooking Method,Distiller's Grains,and Vitamin E Supplementation on the Vitamin Content of Value Cuts from Beef Steers Fed Wet Distiller's Grains and Solubles and Supplemental Vitamin E

ABSTRACT: Vitamin E, thiamin, riboflavin, niacin, vitamin B₆, and vitamin B₁₂ concentrations of flat iron steaks and petite tenders from steers fed finishing rations containing 0% and 40% corn wet distiller's grains and solubles (WDGS) with and without supplemental vitamin E were determined. Feeding treatment groups were: 0% WDGS with basal vitamin E, 0% WDGS with supplemental vitamin E (500 IU daily), 40% WDGS with basal vitamin E, and 40% WDGS and supplemental vitamin E. Cattle can be fed 40% WDGS diets more economically than corn diets. The incorporation of 40% WDGS, with and without vitamin E, was hypothesized to have little effect on the vitamin concentrations of these value meat cuts. Flat iron steaks and petite tenders were broiled and/or grilled to 70 °C internal temperature. Mean cooking yields ranged from 68.7% to 78.2%. The majority of the vitamin concentrations of broiled and of grilled meat were significantly different (P < 0.05) from that of raw meat. Vitamin E concentrations of raw and cooked meat from steers that received supplemental vitamin E were significantly higher (P < 0.05) than those fed basal vitamin E. Significant differences in thiamin, riboflavin, vitamin B₆, and vitamin B₁₂ concentrations in raw flat iron steaks and in vitamin B₆ in raw petite tenders were observed by WDGS. Thiamin, vitamin B₆, and vitamin B₁₂ concentrations of broiled flat iron steaks were significantly different (P < 0.05) than grilled. A few differences in vitamin concentrations of the flat iron steaks and petite tenders were observed by WDGS, vitamin E supplementation, and cooking treatments, but most of the vitamin concentrations were statistically similar.     

The analysis of vitamin B12 in milk and infant formula: A review

Vitamin B₁₂ plays a vital role in human metabolism and is an essential vitamin obtained predominantly from food of animal origin. Amongst all animal products, naturally occurring vitamin B₁₂ in milk has the highest bioavailability and dairy products are a broad-access source, especially for vegetarian individuals. The dairy industry requires an accurate and highly sensitive detection method for vitamin B₁₂, however, the extremely low concentration and instability of vitamin B₁₂ creates challenges in analysis. This review discusses the application of modern instrumental techniques for analysis of vitamin B₁₂ in milk as well as a variety of sample preparations, together with their respective advantages and drawbacks.     

Effects of fermentation time and storage on the water‐soluble vitamin contents of tarhana

Changes in water‐soluble vitamins were investigated in tarhana dough during fermentation and in tarhana stored under a variety of conditions for up to 6 months. The contents of riboflavin (P < 0.01), thiamin, niacin, vitamin B₆ and folic acid (P < 0.05) increased significantly during fermentation. The thiamin, riboflavin, niacin, vitamin B₆ and folic acid contents of tarhana dough were 4.80, 1.85, 7.68, 15.30 and 2.25 mg kg^?1 respectively at the beginning of fermentation and increased to 10.15, 5.15, 24.04, 19.35 and 6.10 mg kg^?1 respectively at the end of fermentation. Thiamin, riboflavin and folic acid contents were significantly affected (P < 0.01) by storage type and period, but niacin and vitamin B₆ contents were not affected (P > 0.05). The water‐soluble vitamin contents of tarhana stored wet were higher than those of tarhana stored dry. Drying resulted in a 24% reduction in riboflavin content and an 86% reduction in folic acid content. Copyright © 2007 Society of Chemical Industry