Comparative study on pressure and temperature stability of 5-methyltetrahydrofolic acid in model systems and in food products

A comparative study on the pressure and temperature stability of 5-methyltetrahydrofolic acid (5-CH(3)-H(4)folate) was performed in model/buffer systems and food products (i.e., orange juice, kiwi puree, carrot juice, and asparagus). Effects of pH and ascorbic acid (0.5 mg/g) on 5-CH(3)-H(4)folate stability in buffer systems were studied on a kinetic basis at different temperatures (from 65 to 160 degrees C) and different pressure/temperature combinations (from 100 to 700 MPa/from 20 to 65 degrees C). These studies showed that (i) the degradation of 5-CH(3)-H(4)folate in all model systems could be described by first-order reaction kinetics, (ii) the thermostability of 5-CH(3)-H(4)folate was enhanced by increasing pH up to 7, (iii) 5-CH(3)-H(4)folate was relatively pressure stable at temperatures lower than 40 degrees C, and (iv) ascorbic acid enhanced both the thermo- and barostabilities of 5-CH(3)-H(4)folate. In food products, temperature and pressure stabilities of 5-CH(3)-H(4)folate were studied at different temperatures (70-120 degrees C) and different pressure/temperature combinations (from 50 to 200 MPa/25 degrees C and 500 MPa/60 degrees C). 5-CH(3)-H(4)folate in orange juice and kiwi puree was relatively temperature (up to 120 degrees C) and pressure (up to 500 MPa/60 degrees C) stable in contrast to carrot juice and asparagus. Addition of ascorbic acid (0.5 mg/g) in carrot juice resulted in a remarkable protective effect on pressure (500 MPa/60 degrees C/40 min) and temperature degradation (120 degrees C/40 min) of 5-CH(3)-H(4)folate.     

Pressure and temperature stability of 5-methyltetrahydrofolic acid: a kinetic study

Detailed kinetic studies of [6S] and [6RS] 5-methyltetrahydrofolic acid (5-CH3-H4folate) degradation during thermal (from 60 to 90 degrees C) and high pressure/thermal (from 30 to 45 degrees C; from 200 to 700 MPa) treatments were carried out. The results confirmed that the temperature and pressure induced degradation kinetics of [6S] 5-CH3-H4folate were identical (within 95% confidence interval) with those of [6RS] 5-CH3-H4folate. Under equal processing conditions, the estimated degradation rate constants (k), activation energy (E(a)), and activation volume (V(a)) values of [6S] and [6RS] 5-CH3-H4folate were the same (95% confidence interval). The modified thermodynamic model proposed by Nguyen and co-workers (J. Agric. Food Chem. 2003, 51, 3352-3357) to describe the pressure and temperature dependence of the rate constant for folate degradation was reevaluated.     

Changes of folate and other potential health-promoting phytochemicals in legume seeds as affected by germination

Folate deficiency associated with low dietary intake is a well-documented public health problem, resulting in serious health and socioeconomic burdens. Therefore, optimization of the germination process of different cultivars of legume seeds in relation to the content and composition of folate, vitamin C, and total phenolics and total antioxidant capacity was carried out to maximize the health-promoting properties. The content and composition of folate, vitamin C, and total phenolic and total antioxidant capacities varied between species, among cultivars, and with germination time. During germination, total folate content was maximum at 815.2 μg/100 g fresh weight in soybean sprout and at 675.4 μg/100 g fresh weight in mungbean sprout on the fourth day, which were equivalent to, respectively, 3.5- and 3.9-fold increases in the seed's content, and total folate content strongly decreased thereafter. 5-CH(3)-H(4)folate was the most abundant folate species in legume sprouts and reached a maximum on the fourth day. Vitamin C was not detected in raw seeds, and its content increased sharply in soybean and mungbean sprouts and reached a maximum at the fourth day of germination (29 and 27.7 mg/100 g fresh weight, respectively). Germination of soybean and mungbean for 4 days provided the largest amount of total folate as well as the more stable species 5-CH(3)-H(4)folate and also brought about large amounts of vitamin C and total phenolics and substantial antioxidant capacities.     

Natural variation of folate content and composition in spinach (Spinacia oleracea) germplasm

Breeding to increase folate levels in edible parts of plants, termed folate biofortification, is an economical approach to fight against folate deficiency in humans, especially in the developing world. Germplasm with elevated folates are a useful genetic source for both breeding and direct use. Spinach is one of the well-know vegetables that contains a relatively high amount of folate. Currently, little is known about how much folate, and their composition varies in different spinach accessions. The aim of this study was to investigate natural variation in the folate content and composition of spinach genotypes grown under controlled environmental conditions. The folate content and composition in 67 spinach accessions were collected from the United States Department of Agriculture (USDA) and Asian Vegetable Research and Development Center (AVRDC) germplasm collections according to their origin, grown under control conditions to screen for natural diversity. Folates were extracted by a monoenzyme treatment and analyzed by a validated liquid chromatography (LC) method. The total folate content ranged from 54.1 to 173.2 μg/100 g of fresh weight, with 3.2-fold variation, and was accession-dependent. Four spinach accessions (PI 499372, NSL 6095, PI 261787, and TOT7337-B) have been identified as enriched folate content over 150 μg/100 g of fresh weight. The folate forms found were H(4)-folate, 5-CH(3)-H(4)-folate, and 5-HCO-H(4)-folate, and 10-CHO-folic acid also varied among different accessions and was responsible for variation in the total folate content. The major folate vitamer was represented by 5-CH(3)-H(4)-folate, which on average accounted for up to 52% of the total folate pool. The large variation in the total folate content and composition in diverse spinach accessions demonstrates the great genetic potential of diverse genotypes to be exploited by plant breeders.     

Folate content in strawberries (Fragaria x ananassa): effects of cultivar,ripeness, year of harvest,storage, and commercial processing

Folate concentrations in strawberries and folate retention during storage and commercial processing of strawberries were investigated. No previous study has focused on the effects of cultivar, ripeness, and year of harvest of strawberries with respect to the folate content. This study showed the folate concentration in strawberries to significantly depend on all of these different factors. Total folate was quantified using a modified and validated radioprotein-binding assay with external calibration (5-CH(3)-H(4)folate). Folate content in 13 different strawberry cultivars varied from 335 microg/100 g of dry matter (DM) for cv. Senga Sengana to 644 microg/100 g of DM for cv. Elsanta. Swedish harvests from 1999 and 2001 yielded higher folate concentrations than did the harvest from 2000, and the grade of ripeness affected the folate content in strawberries. This study indicated high folate retention in intact berries during storage until 3 or 9 days at 4 degrees C (71-99%) and also in most tested commercial products (79-103%). On the basis of these data fresh strawberries as well as processed strawberry products are recommended to be good folate sources. For instance, 250 g (fresh weight) of strawberries ( approximately 125 microg of folate) supplies approximately 50% of the recommended daily folate intake in various European countries (200-300 microg/day) or 30% of the U.S. recommendation (400 microg/day).     

Effect of Baking Method and Fermentation on Folate Content of Rye and Wheat Breads

The effect of baking method on folates of rye and wheat breads, as well as the effect of sourdough fermentation of rye, were examined. Sourdough fermentations were performed both with and without added yeast, and samples were taken throughout the baking process. Samples were analyzed microbiologically for their total folate content after trienzyme extraction. Individual folate vitamers were determined by HPLC after affinity chromatographic purification. The lowest folate contents for both rye and wheat breads were found from breads baked without added yeast. Total folate content increased considerably during sourdough fermentation due to increased amounts of 10‐HCO‐H₂folate, 5‐CH₃‐H₄folate, and 5‐HCO‐H₄folate. Baker's yeast contributed markedly to the final folate content of bread by synthesizing folates during fermentation. Proofing did not influence total folate content but changes in vitamer distribution were observed. Folate losses in baking were ≈25%. The variety of sourdoughs and baking processes obviously lead to great variation in folate content of rye breads. The possibilities to enhance natural folate content of rye bread by improving folate retention in technological processes and by screening and combining suitable yeasts and lactic acid bacteria should be further investigated.     

Development of a simplified method for the determination of folates in baker's yeast by HPLC with ultraviolet and fluorescence detection

A simplified HPLC method for rapid determination of folates in yeast with ultraviolet and fluorescence detection without sample purification has been developed. By use of the column Aquasil C(18), specially designed for polar analytes, and gradient elution, it was possible to separate and determine five folate derivatives: tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate with fluorescence detection, and 10-formylfolic acid and folic acid with ultraviolet detection. The sample preparation required only a small amount of dry yeast (25-50 mg) and included an extraction of folates by heat treatment and deconjugation of folate polyglutamates to monoglutamates with the use of rat serum conjugase. Validation involved investigation of matrix effects, determination of recovery by standard addition method, repeatability, and stability tests. The dominating folate forms in commercial dry baker's yeast were found to be tetrahydrafolate and 5-methyltetrahydrofolate with a total folate content of 2890 microg/100 g (63.4 nmol/g). The simplicity of the method makes it suitable for folate screening studies of different yeast strains.     

HPLC Determination of Stability and Distribution of Added Folic Acid and Some Endogenous Folates During Breadmaking

Bread flour was spiked with folic acid (1.40 mg/lb or 3.08 μg/g of flour) and processed into bread by the sponge and dough method. Changes that occurred to added folic acid and endogenous folate contents through different processing stages, including sponge formation, proofing, and baking, were assessed by reversed‐phase ion‐pair HPLC combined with UV and fluorometric detection. Sample extraction required α‐amylase and rat plasma deconjugase digestion, and sample preparation required purification by solid‐phase extraction. Added folic acid was measured by monitoring UV absorption at 280 nm. Four selected forms of endogenous folates including tetrahydrofolate (THF), 5‐formyl‐THF, 10‐formylfolate, and 5‐methyl‐THF were identified and quantified throughout the bread processing using a fluorescence excitation wavelength of 290 nm and emission wavelength of 350 or 450 nm. Data indicate a relatively good stability of added folic acid and native folates to the baking process, and increased endogenous folate contents in dough and bread as compared with the flour from which they were made.     

Protein utilization during soybean tempe fermentation.

The aim was to identify to what extent proteins were utilized during the fermentation of bacteria-free tempe prepared with acidified soybean cotyledons and Rhizopus oligosporus NRRL 2710 at 30 degrees C. Dry matter declined continuously during the fermentation to 980 g/(kg of initial dry cotyledons) at 28 h, 910 g at 46 h (when the tempe was judged mature), and 835 g at 72 h. The decrease in dry matter was due mainly to reduction in crude lipid, amounting to 65 g/(kg of initial dry cotyledons) at 46 h and 135 g/(kg of initial dry cotyledons) at 72 h and representing approximately 70% and 80%, respectively, of the total dry matter loss. Protein oxidation (estimated from ammonia production) was 5 g at 28 h,10 g at 46 h, and 20 g/(kg of initial dry cotyledons) at 72 h. The total amount of soy protein hydrolyzed, including that incorporated into mold biomass, was estimated to be 80 g/(kg of initial dry cotyledons) at 28 h incubation, 95 g at 46 h, and 100 g at 72 h. The hydrolyzed protein at 46 h represented 25% of the initial protein. Of this hydrolyzed protein, it is suggested that approximately 65% remained in the tempe as amino acids and peptides, 25% was assimilated into mold biomass, and 10% was oxidized.     

Affinity and rate constants for interactions of bovine folate-binding protein and folate derivatives determined by optical biosensor technology. Effect of stereoselectivity

The interactions between bovine folate-binding protein (FBP) and different folate derivatives in pure diastereoisomeric forms were studied at pH 7.4 by a surface plasmon resonance technology (Biacore). The results show that folic acid had the most rapid association rate (k(a) = 1.0 x 10(6) M(-)(1) s(-)(1)), whereas (6S)-5-HCO-5,6,7,8-tetrahydrofolic acid had the most rapid dissociation rate (k(d) = 3.2 x l0(-)(3) s(-)(1)). The equilibrium dissociation constant (K(D)), calculated from the quotient of k(d)/k(a), showed that the two forms of folates not occurring in nature, that is, folic acid and (6R)-5-CH(3)-5,6,7,8-tetrahydrofolic acid, had the highest affinities for FBP, 20 and 160 pmol/L, respectively. The results thus show that there were great differences in the interactions between folate-binding protein and the major forms of folate derivatives. The nutritional implications of these differences are discussed.     

Folate in wheat genotypes in the HEALTHGRAIN Diversity Screen

As part of the diversity screen of the HEALTHGRAIN project, the total folate contents of bread wheat (130 winter and 20 spring wheat genotypes), durum wheat (10 genotypes), earlier cultivated diploid einkorn and tetraploid emmer wheat (5 genotypes of each), and spelt (5 genotypes), grown in the same location in a controlled manner, were determined by a microbiological assay. The total folate contents ranged from 364 to 774 ng/g of dm in winter wheat and from 323 to 741 ng/g of dm in spring wheat, thus showing a marked variation. The highest mean for total folate content was measured in the durum wheat genotypes, whereas the earlier cultivated diploid and tetraploid wheat genotypes and spelt were shown to possess comparable or even higher folate contents than bread wheat. HPLC analysis of selected genotypes showed that 5-formyltetrahydrofolate was the major vitamer. The data provide a basis for breeding wheat genotypes with improved folate content.     

Lipoxygenase from banana leaf: purification and characterization of an enzyme that catalyzes linoleic acid oxygenation at the 9-position

The objective of the present study was to purify and characterize the lipoxygenase (LOX) from banana leaf (Giant Cavendishii, AAA), an unutilized bioresource. LOX was extracted, isolated, and purified 327-fold using 25-50% saturation of ammonium sulfate fractionation, hydroxyapatite column separation, and gel filtration on Superdex 200. The molecular mass of the purified LOX was 85 kDa, K(m) was 0.15 mM, and V(max) was 2.4 microM/min.mg using linoleic acid as substrate. Triton X-100 was required in the extraction medium; otherwise, no LOX activity was detected. LOX activity increased with the concentration of Triton X-100 with an optimum at 0.1%. The optimal pH of the purified LOX from banana leaf was 6.2, and optimal temperature was 40 degrees C. The LOX showed the highest reactivity toward 18:2 followed by 18:3 and 20:4. A very low reaction rate was observed toward 20:5 and 22:6. On the basis of retention time in normal phase HPLC, the products of 18:2 or 18:3 catalyzed by purified LOX were hydroperoxyoctadecadienoic acid or hydroperoxyoctadecatrienoic acid. It seems that 9-LOX is the predominant enzyme in banana leaf. Banada leaf dried at 110 degrees C for 2 h developed algal aroma. Banana leaf extract stored at 10 degrees C for 12 h formed an oolong tea-like flavor. Banana leaf extract reacted with 18:2 or soybean oil pretreated with bacterial lipase produced green and melon-like aroma, whereas the same reaction with 18:3 produced a sweet, fruity, cucumber-like flavor note.     

Synthesis and fungicidal activity of novel 4,4'-bis(2' '-aryl-5' '-methyl/unsubstituted-4' '-oxo-thiazolidin-3' '-yl) bibenzyl

Reduction followed by nitration of benzil I yielded 4,4'-dinitrobibenzyl (III) which by reduction furnished quantitatively and analytically pure 4,4'-diaminobibenzyl (IV) which on condensation with different carbonyl compounds gave 4,4'-bis (benzylideneamino) bibenzyls (Va-f). Compounds (Va-f) on cycloaddition with mercaptoacetic acid/2-mercaptopropionic acid yielded the corresponding 4-oxothiazolidin-3-yl bibenzyls (VIa-l). The compounds VIg-l have two chiral centers in each thiazolidinone moiety so two diastereomers are possible, but on crystallization and repeated chromatography, one diastereomer was obtained. The absolute configuration of the diastereomer was tentatively assigned on the basis of (1)H NMR spectra. (1)H NMR spectra of the product showed a distinct doublet at delta 1.22 for C(5)-CH(3) of thiazolidinone ring (22, 23) and a distinct quartet at delta 4.20 for the C(5)-H proton. Similarly, the C(2) proton showed an independent singlet at delta 5.95, so the diastereomers obtained were assigned trans configuration. Compounds Va-f and VIa-l were evaluated in vitro for their fungitoxicities against Fusarium oxysporium and Penicillium citrinum. All the compounds were found to be antifungal active. Some of the compounds displayed activities comparable with that of the commercial fungicide Dithane M-45. Structure-activity relationships for the screened compounds are discussed.     

Syntheses of labeled vitamers of folic acid to be used as internal standards in stable isotope dilution assays

[2H4]Folic acid was synthesized by deuterating p-aminobenzoic acid, which was then coupled to glutamic acid and 6-formylpterin. Using [2H4]folic acid as starting component enabled the preparation of labeled vitamers tetrahydrofolate, 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, and 10-formylfolate which were characterized by electrospray mass spectrometry and collision-induced dissociation. The mass spectrometric studies confirmed that the compounds could be used as internal standards in stable isotope dilution assays.     

Isoflavone transformation during soybean koji preparation and subsequent miso fermentation supplemented with ethanol and NaCl

Soybeans were soaked with water for 4 h, steam-cooked, inoculated with the conidia of Aspergillus oryzae, and incubated for 3 days for koji preparation. The koji was then mixed with water-soaked and steam-cooked soybeans (1:2, w/w), ground into paste, and supplemented with 15% ethanol and 12.5% NaCl or 3% ethanol and 6% NaCl for miso fermentation at 30 degrees C. Daidzin, genistin, daidzein, and genistein contents were extracted from the lyophilized and pulverized soybean powder or from the miso homogenate by a developed one-tube procedure and analyzed with an HPLC. After water soaking, daidzein and genistein contents increased markedly, whereas daidzin and genistin contents decreased. Further increases of daidzein and genistein contents and decreases of daidzin and genistin contents were observed after koji mold growth. During fermentation, fungal and lactic acid bacterial (LAB) growth in the miso products was inhibited, whereas soluble protein contents increased much more rapidly in the low-salt miso products supplemented with 3% ethanol and 6% NaCl than the other products. When the 4- and 8-week-fermented miso products were cooked with tofu for sensory evaluation, flavor ratings of the low-salt products were higher than that of a popular commercial product. In both products, the most daidzins and genistins were hydrolyzed after 4 weeks of fermentation. The hydrolytic enzymes contributing to isoflavone transformation originated from soybeans after water soaking and from koji with mold growth. It was of merit that the low-salt fermented products were fairly acceptable in flavor rating and rich in daidzein and genistein contents after 4 weeks of fermentation.     

Identification of conjugated linoleic acids in hydrogenated soybean oil by silver ion-impregnated HPLC and gas chromatography-ion impacted mass spectrometry of their 4,4-dimethyloxazoline derivatives

Hydrogenated soybean oil was obtained after 10 min of hydrogenation with 0.5% selective type Ni catalyst at 230 degrees C, a hydrogenation pressure of 0.049 MPa, and an agitation rate of 300 rpm. The conjugated linoleic acid isomers in the hydrogenated soybean oil were isolated by using a silver ion-impregnated HPLC. Gas chromatography-mass spectrometry of 4,4-dimethyloxazoline (DMOX) derivatives of the isolated conjugated linoleic acid isomers were carried out for the identification of their chemical structures. By interpreting the mass spectra of the DMOX derivatives of conjugated linoleic acid isomers isolated by silver ion-impregnated HPLC, 20 different conjugated linoleic acid isomers present in hydrogenated soybean oil were identified. This is the first report for the mass spectrometric identification of the conjugated linoleic acid isomers present in hydrogenated vegetable oil.     

"Untypical aging off-flavor" in wine: synthesis of potential degradation compounds of indole-3-acetic acid and kynurenine and their evaluation as precursors of 2-aminoacetophenone

Kynurenine (1) and indole-3-acetic acid (2) are considered as potential precursors of 2-aminoacetophenone (3), which is regarded to be the aroma impact compound causing an "untypical aging off-flavor" (UTA) in Vitis vinifera wines. The mechanism of the formation of 3 was studied using model fermentation and model sulfuration media spiked with 1 or 2 as potential precursors. Possible degradation products such as kynurenamine (4) and kynurenic acid (5), or skatole (6), 2-oxoskatole (7), 2-formamidoacetophenone (8), 2-oxindole-3-acetic acid (9), and 3-(2-formylaminophenyl)-3-oxopropionic acid (10) were evaluated by HPLC-UV of the fermentation and sulfuration media and comparison with synthesized 7, 8, 9, and 10. The synthesis of the possible precursor 4-(2-aminophenyl)-2,4-dioxobutanoic acid (11), a proposed metabolite of 1 failed because a spontaneous cyclization yields 5 and N-oxo-kynurenic acid (12), but not 11. It could be shown that the formation of 3 is triggered by an oxidative degradation of 2 after sulfuration with potassium bisulfite via the intermediates 10 and 8. However, no formation of 3 occurred during sulfuration of a model wine spiked with 1 or during fermentation of a model must spiked with 1 or 2.     

Improved folate extraction and tracing deconjugation efficiency by dual label isotope dilution assays in foods

A dual label stable isotope dilution assay was developed to trace the deconjugation efficiency of polyglutamic folate vitamers converted to their monoglutamic analogues. For this purpose, [(13)C(5)]-pteroylheptaglutamate was synthesized and added during extraction of foods as a tracer isotopologue along with [(2)H(4)]-5-methyltetrahydrofolate, [(2)H(4)]-5-formyltetrahydrofolate, [(2)H(4)]-tetrahydrofolate, [(2)H(4)]-10-formylfolate, and [(2)H(4)]-folic acid. The [(2)H(4)]-labeled folates were used as internal standards for the monoglutamates. Deconjugation converted the addition tracer [(13)C(5)]-pteroylheptaglutamate to the detection tracer [(13)C(5)]-folic acid, which was quantified along with unlabeled folic acid using [(2)H(4)]-folic acid as the internal standard. LC-MS/MS enabled the unequivocal differentiation of the three isotopologues. This tracing was used to optimize deconjugation efficiency, which was achieved by using 4-morpholineethanesulfonic acid buffer for extraction at pH 5.0 . The optimized assay revealed limits of detection for the folate vitamers ranging between 2.0 and 5.6 pmol per assay (equivalent to 2.2-6.6 μg/100 g dry mass), recoveries ranging between 98 and 105% and relative standard deviations in inter-assay precision ranging between 2 and 6%. The assay was applied to quantitate folates in spinach, beans, cheeses, bread, wheat germs, and yeast .     

Influence of the application of three different elicitors on soybean plants on the concentrations of several isoflavones in soybean seeds

Soybean [Glycine max (L.) Merr.] is a rich source of isoflavones that are often affected by biotic and abiotic factors. The objectives of this study were to evaluate the effect of various concentrations of three natural elicitors applied at different soybean growth stages on isoflavone content and to compare the efficiency of several solvent systems in isoflavone extraction and quantification. The isoflavones extracted from R96-3444 soybean using eight solvent systems were separated, identified, and quantified by a high-performance liquid chromatography (HPLC) procedure. The soybean plants were sprayed with salicylic acid, methyl salicylate, or ethyl acetate at 0, 10(-6), 10(-3), and 10(-1) M at R1 (blooming) or R4 (full pods) growth stage. Results showed that 10(-3) M ethyl acetate sprayed at the R1 stage significantly increased total isoflavone content and the levels of some individual isoflavones in soybean seeds. With all the elicitors that were tested, concentration was a more important factor than application time with respect to isoflavone content with lower concentrations being more effective on most isoflavones. A 53% acetonitrile solvent system was the best solvent system for extracting total isoflavone, malonyl glucosides, genistein, glycitin, genistin, acetyl-daidzin, and acetyl-genistin. The results of this study will be useful for increasing the isoflavone content in desirable soybean varieties and improving isoflavone concentration during extraction.     

1H NMR, a rapid method to monitor organic acids during cupuassu (Theobroma grandiflorum Spreng) processing

The development of an analytical method using 1H nuclear magnetic resonance (1H NMR) spectrometry to monitor cupuassu (Theobroma grandiflorum Spreng) bean fermentation, drying, and roasting processes is reported. The analysis of organic acids and alcohols of crude water extracts of cupuassu ground kernels were monitored by HPLC and 1H NMR spectroscopy. The residual protein signals caused deleterious effects on acid and alcohol quantifications. Therefore, the analytical procedures were optimized by sample cleanup and water suppression pulse sequences in order to obtain compatible data using HPLC and 1H NMR. The quantification of lactic acid, acetic acid, and 2,3-butanediol by NMR is 5- to 10-fold faster than by HPLC, with the advantage of providing the identification of several chemical species in a single experiment. Application of these analytical conditions to some cupuassu samples revealed that this methodology can be applied to the quality profiles of fermentation and roasting processes.