Folate In Vegetables Research Articles

Optimization and Application of FL-HPLC for Folates Analysis in 20 Species of Mediterranean Wild Vegetables

Folic acid and folates (vitamin B9) are essential for human metabolism. Their nutritional importance mainly lies in the prevention of neural tube formation defects, and other degenerative diseases. The determination of folates in food matrices is a very complex task due to the large number of chemical compounds included under the definition of folates. In the present study, an HPLC-fluorescence detection (HPLC-FL) methodology was optimized and validated to characterize total folates in vegetables. Its application to Mediterranean wild vegetables, provide new data on folates content on these species, scarcely studied so far. The highest folates contents were found in the leaves of Rumex pulcher L., Beta maritima L. and Anchusa azurea Mill. (506, 302 and 278 μg/100 g, respectively), as well as in the tender leaves and stems of Foeniculum vulgare Mill. and Silene vulgaris (Moench.) Garcke (272 and 268 μg/100 g, respectively). These data show that the studied wild vegetables are mostly a very good source of folates, compared to other folate-rich plants, such as spinach, parsley or chard. These under-utilized plants deserve attention for their potential value in current diets, as they are a good alternative for the diversification of our vegetable intake and represent an important added value as rich sources of vitamin B9.

Determination of folates in vegetables and their retention during boiling

HPLC method for 5-methyltetrahydrofolate (5-MTHF) determination in vegetables was optimised for the folate release from the food matrix. Enzymatic hydrolyses using the subsequent addition of α-amylase, protease, and conjugase from hog kidney, or their combinations, were tested. The highest release values were obtained with the application of enzymes α-amylase and conjugase, amounting to 112.4–127.0% of the values obtained in the processing with sole conjugase. The simultaneous addition of both enzymes and the incubation at pH 4.9 did not suppress the release of folates. Spinach, Chinese cabbage, lettuce, cauliflower, and broccoli contained more than 50 μg of 5-MTHF/100g, whereas less than 25 μg/100 g was found in potatoes, carrot, white cabbage, green and yellow pepper. Individual vegetables differed in the folate retention during their boiling under constant conditions. The highest retention was found in Brussels sprouts, cauliflower, and broccoli. After 8 min boiling more than 75% of the initial amount of 5-MTHF remained in these vegetables. Lower values of 5-MTHF retention, between 37% and 52% of their initial content, were found in spinach, savoy cabbage, and carrot.

Optimization of the Trienzyme Extraction for the Microbiological Assay of Folate in Vegetables

Response surface methodology was applied to optimize the trienzyme digestion for the extraction of folate from vegetables. Trienzyme extraction is a combined enzymatic digestion by protease, alpha-amylase, and conjugase (gamma-glutamyl hydrolase) to liberate the carbohydrate and protein-bound folates from food matrices for total folate analysis. It is the extraction method used in AOAC Official Method 2004.05 for assay of total folate in cereal grain products. Certified reference material (CRM) 485 mixed vegetables was used to represent the matrix of vegetables. Regression and ridge analysis were performed by statistical analysis software. The predicted second-order polynomial model was adequate (R2 = 0.947), without significant lack of fit (p > 0.1). Both protease and alpha-amylase have significant effects on the extraction. Ridge analysis gave an optimum trienzyme digestion time: Pronase, 1.5 h; alpha-amylase, 1.5 h; and conjugase, 3 h. The experimental value for CRM 485 under this optimum digestion was close to the predicted value from the model, confirming the validity and adequacy of the model. The optimized trienzyme digestion condition was applied to five vegetables and yielded higher folate levels than the trienzyme digestion parameters employed in AOAC Official Method 2004.05.

Retention of folates in cooked, stored and reheated peas, broccoli and potatoes for use in modern large-scale service systems

This study aimed to evaluate retention of folate in vegetables caused by different processes used in modern large-scale service systems and the food industry. The concentration of folates present in raw samples of peas, broccoli and potatoes was measured during different cooking methods, warm and cold holding and reheating. The main folate forms in vegetables, tetrahydrofolates and 5-methyltetrahydrofolates, were analysed using a validated high-performance liquid chromatography (HPLC) method. This study shows the following decreasing order in folate retention, on DM basis, compared to raw potatoes during heat-processing: sous-vide (103%), boiling (72–59% (unpeeled and peeled)) and oven-baking (63%) and compared to raw green peas during heat processing: boiling (77%), microwaving (75%), steam boiling (73%) and blanching (71%). However, only blanching of peas, boiling of potatoes and oven-baking of unpeeled potatoes caused significant reduction. Storage at various temperatures and length of times followed by reheating caused no further significant losses of total folate.

Determination of folate contents in some Australian vegetables

The undeconjugated and total folate contents of 22 vegetables commonly available in Australia were assayed with Lactobacillus casei after preliminary digestion with chicken pancreas. The effect of conjugate type and extraction technique on the total folate content of two of the vegetables was also investigated. In two test samples, spinach and Chinese broccoli, deconjugation with chicken pancreas gave slightly higher, but statistically not different ( P<0.05), folate values compared to human plasma. Tri-enzyme extraction gave lower folate value compared to single-enzyme extraction, the result was significant ( P<0.05) only in Chinese broccoli. Single-enzyme extraction using chicken pancreas was therefore used for extraction of folate in all vegetables. The range of undeconjugated and total folate in vegetables was 27–322 and 68–425 μg total folate/100 g (wet weight), respectively. The average undeconjugated to total folate ratio in 22 vegetables was 0.60, ranging from 0.19 to 0.89. Chinese flowering cabbage contained the highest level of total folate at 425 μg /100 g. Among the rest of the 21 vegetables, four vegetables contained more than 300 μg, five contained 200–300 μg, 10 contained 100–200 μg, and two contained less than 100 μg folate/100 g.

HPLC determination of folates in raw and processed beetroots

A sensitive HPLC method with fluorescence detection and gradient elution has been developed for the determination of folates in vegetables. The method involved extraction of folates from food matrix by heat treatment, deconjugation of folate polyglutamates to monoglutamates by incubation with hog kidney conjugase and purification of food extracts by solid-phase extraction with strong-anion exchange cartridges. The chromatographic separation of folates was achieved on Zorbax SB C 8 column, which was found to be superior over conventional C 18 column in terms of selectivity and sensitivity. Validation of the method included linearity tests, the addition of standard folates for the determination of recovery, repeatability and stability tests. The method developed was applied to analysis of raw and processed beetroots; 5-methyltetrahydrofolate was found to be the main folate form in beetroots. Cultivar differences and growing conditions were found to have a pronounced effect on the folate content in beetroots. Processing resulted in considerable losses of folates, whereas losses during storage appeared to be moderate.

Influence of processing on total, monoglutamate and polyglutamate folate contents of leeks, cauliflower, and green beans.

Bioavailability of dietary folate might be impaired by the polyglutamate chain to which approximately 70% of dietary folates are bound. This chain must be removed enzymatically in the intestine before folate is absorbed as a monoglutamate. To increase formation of monoglutamate folate in vegetables, the vegetables were subjected to various processing treatments. Treatments included freezing (-18 degrees C, 16 h) and thawing (4 degrees C, 24 h) and hydrostatic high-pressure treatment (200 MPa, 5 min). Both freezing/thawing and high-pressure treatment increased the proportion of folate in the monoglutamate form in leeks, cauliflower, and green beans 2-3-fold. However, loss of total folate after these treatments was >55%. It is concluded that conversion of folate polyglutamate to the monoglutamate form in vegetables is possible by certain processing treatments. Potentially this could lead to vegetables with higher folate bioavailability. However, to prevent folate loss into processing water, processing in a closed system should be applied.

A Validated Liquid Chromatographic Method for Determining Folates in Vegetables, Milk Powder, Liver, and Flour

A liquid chromatographic (LC) method was elaborated for determining folates in foods. Folates were extracted by homogenizing in buffer and heat treatment. A portion was incubated with an enzyme preparation containing conjugase, amylase, and protease. After purification by affinity chromatography, folate monoglutamates were determined by reversed-phase LC with fluorescence and diode array detection. Gradient elution with phosphate buffer and acetonitrile was used to separate vitamers. The most abundant folate forms naturally present in foods were detected, including tetrahydrofolic acid, 5-methyltetrahydrofolic acid, and 5-formyltetrahydrofolic acid. 10-Formylfolic acid could be detected by applying a second fluorescence detector. Folic acid, used for fortification, might also be quantitated with this system. The difference between folate concentrations in sample extracts, with and without treatment of conjugase, is a measure of the quantity of polyglutamates in the food matrixes. An additional treatment with conjugase, amylase, and protease reflects the amount of matrix-bound folates. The LC system gave a linear response over the range 0-100 ng/mL. Detection limit for these compounds were 7 pg/mL for tetrahydrofolic acid and 5-methyltetrahydrofolic acid and 59 pg/mL for 10-formylfolic acid (signal-to-noise ratio > or = 3) when 100 microL was injected. Detection limits for 5-formyltetrahydrofolic acid and folic acid were 1 ng/mL. Repeatability relative standard deviation values for separate folates in 3 candidate Certified Reference Materials (CRMs)--mixed vegetables (CRM 485), pig liver (CRM 487), and whole-meal flour (CRM 121)--and a Certified Reference Material milk powder (CRM 421) varied from 3.3 to 21.0% for the concentration range 1.8-1440 micrograms/100 g. Recoveries ranged from 73 to 109%. Use of amylase and protease was advantageous. Use of a commercially available folate-binding protein for cleanup saved time and money and was effective. Results for 5-methyltetrahydrofolic acid were in good agreement with results obtained with other LC methods. Results for total folates were lower than results obtained with microbiological methods.