Folate composition of selected food samples Lupin flour

Our study obtained a total folate content of 1058 ng/g in the flour from lupin seeds grown in 2011, a result which is similar to the value (1130 ng/g) reported by Mäkelä (2012) who used samples from the same harvest. However, folate contents of the blue lupin seeds from the 2010 harvest were found to be much higher; and the contents differed significantly between growing locations, ranging from 3420 ng/g (Mikkeli) to 3990 ng/g (Helsinki), but were not affected by the cultivars (Lizarazo et al. 2011; Rekola 2011). According to the USDA nutrient data, raw lupin seeds contain 3550 ng folic acid/g. Hence, the folate contents of lupin could be greatly influenced by extrinsic factors such as growing conditions and the harvest year.

Meanwhile, Rekola (2011) identified the folates in the lupin seed of Boruta harvested in Helsinki, which constituted 5-CH3-THF (60%), 5-CHO-THF (29%) and minor components including THF, 5,10-CH⁺-THF, 10-CHO-FA and folic acid, a profile which was in an

agreement with our findings. However, they found same folyl components with a different distribution in the same cultivar grown in Mikkeli. Thus, the folate profile of a certain cultivar seemed to be less affected by the harvest year in spite of great variations in the total folate content.

In our study, the 5-CH3-THF pool of lupin flour was highly polyglutamylated at 87%, with pentaglutamate as the predominated form. In plants and animals, folyl pentaglutamates occupied a large proportion of the folate pools (Pfeiffer and Gregory 1996; Scott et al.

2000; Matella et al. 2005; Becker et al. 2012). The major folates in pea cotyledons, leaves and chloroplasts were found to be tetra- and pentaglutamyl derivatives (Imeson et al. 1990;

Besson et al. 1993). In lima beans, the majority of folates were pentaglutamates of 5-CHO-THF (47%), 5-CH3-THF (35%), 10-CHO-THF (9%) and THF (9%) (Seyoum and Selhub 1993). In plants, the glutamate chain length of cellular folates is affected by physiological states and nutritional status (Crosti et al. 1993; Cossins 2000), and it regulates the one-carbon metabolism by affecting the intracellular flux of one-one-carbon units among competing pathways (Krumdieck et al. 1992). During germination and developmental stages, the folate pool was dominated by highly glutamylated 5-CH3-THF derivatives, which implied their importance in the largest anabolic flux for methionine synthesis and turnover of S-adenosylmethionine (SAM) (Cossins 2000). Meanwhile, the polyglutamylation degree varies among plant species and tissues (Imeson et al. 1990; Zheng et al. 1992).

Faba bean flour

Faba bean is a folate-rich dietary source, and increasing the consumption of faba beans has been associated with an enhanced folate intake (Bermejo et al. 2009). Our UPLC measurement was about 13% lower than the average content of 1500 ng folates/g determined by Konings et al. (2001), which might be due to the loss during affinity purification and a high LOQ for 10-CHO-DHF. Meanwhile, the folate contents of faba beans showed significant differences between cultivars and sites, ranging from 1500 to 3000 ng/g (Rekola 2011). In addition, the dominancy of pentaglutamyl 5-CH3-THF was also found in faba bean flour as expected.

When comparing our results with literature data, the folate composition of faba bean were found to vary among different sources. Rekola (2011) determined the folate composition of the Kontu variety, which was mainly comprised of 5-CH3-THF (47%), 5,10-CH⁺-THF

(28%), CHO-THF (17%) and THF (4%), and of Tattoo variety where there was 34% 5-CH3-THF, 25% 5-CHO-THF, 23% 10-CHO-FA and 14% 5,10-CH⁺-THF. In a study conducted by Hefni et al. (2010), faba beans contained 960 ng folic acid/g with a folate profile of 160 ng THF/g, 730 ng 5-CH3-THF/g and 100 ng 10-CHO-FA/g. Such discrepancies in the folate profile of faba beans might be attributed to both intrinsic and extrinsic factors. Firstly, the folate distribution in plants is associated with the physicological conditions and/or the folate metabolism in different compartments (Cossins 2000). While 5-CH3-THF is involved in the conversion of homocysteine to methionine; 5-CHO-THF might implicate the biosynthesis of purine and formylmethionyl-tRNA, and 5,10-CH2-THF for the formations of thymidylate and pantothenate (Hanson and Roje 2001). Moreover, during sample preparation stages, the heat treatment and the pHs of extraction buffer and purification eluent could induce non-enzymatic interconversion of various folates (De Brouwer et al. 2007).

Dry yeast

In 1931, yeast or a yeast extract was found their curative effects on tropical macrocytic anaemia in the Indian pregnant patients, which was later found to result from the benefits of the folate components in yeasts (Wills et al. 1931). Our sample is belonged to the common strain used for commercial baker’s yeasts−Saccharomyces cerevisiae−which is regarded as an abundant source of folates. An agreement was found between our results and previous data, which were reported in a range of 10-40 μg folates/g (Witthöft et al.

1999; Patring et al. 2005a; Patring et al. 2009). Hjortmo et al. (2005) studied the concentrations of total folates in 44 yeast strains, which ranged from 40 to 145 μg/g.

According to our data, the folate derivatives in dry yeasts were primarily present in the forms of methylated polyglutamates of which heptaglutamate contributed to the greatest proportion, which was in accordance with previous data. The distribution of 5-CH3 -H4PteGlu derivatives in yeasts varied greatly from 33 to 92% among different strains in the research of Hjortmo (2005), and was above 65% in all strains tested by Roje et al. (2002).

In 1976, Bassett et al. have already estimated that the folate distribution during the growth cycle of yeast extracts was 12-16% for hexa-, 67-71% for hepta-, and 10-13% for octa-glutamates. According to the HPLC analysis conducted by Seyoum and Selhub (1998), yeast folates were comprised of unsubstituted (20%) and methylated (77%) polyglutamates,

with 17% 5-CH3-H4PteGlu6, 70% 5-CH3-H4PteGlu7 and 13% 5-CH3-H4PteGlu8 in the latter cluster.

Owing to both their high folate contents and ability to synthesis folates, baker’s yeasts have been widely reported for their great potential for the folate enhancement in bread (Keagy et al. 1975; Kariluoto et al. 2004; Hjortmo et al. 2005). During fermentation, the folate contents of rye sourdough experienced a considerable increase compared to that of flour, with enrichment of 54% and 128% depending on the flour cultivars (Kariluoto et al.

2004). Thus, by selecting most effective strains, yeast could be a vehicle for the folate enhancement in yeast-containing foods such as bread, kefir, cheese, beer, etc. In addition, the utilisation of yeast tended to modify the folate profile of used flour, primarily leading to a high proportion of 5-CH3-THF in breads (Patring et al. 2009).

4 CONCLUSIONS

To our knowledge, this was the first UPLC-FLR/PDA method enabling simultaneous determination of intact polyglutamyl 5-methyltetrahydrofolates and common monoglutamyl folates including THF, 5-CHO-THF, 10-CHO-DHF, 10-CHO-FA, 5,10-CH⁺-THF and folic acid. The chosen UPLC column BEH C18 provided adequate selectivity, specificity and linearity for the analysis of target folate forms without sacrificing peak shapes. The optimised UPLC method enabled fast and sensitive determination of folate polyglutamates in the femtomole level (LOQs ≤ 13.5 fmol/injection) and monoglutamates in the picogram range (LOQs: 6-600 pg/injection). Meanwhile, as an indispensable preparatory step for LC analysis, affinity chromatography (AC) was feasible for the purification of 5-methyl polyglutamates with desirable recoveries, but it resulted in varying losses of the polyglutamates of 5-CHO-THF, depending on the column load and the polyglutamylation degree. In addition, AC was shown to efficiently result in chromatograms devoid of matrix-derived interfering peaks for legume and yeast samples.

Alteration of the sequence of tri-enzyme and purification treatments could significantly affect the total measurable folates and consequent folate composition of the lupin flour.

Our standard procedure¾a simultaneous amylase and conjugase treatment followed by protease treatment and finally AC¾was ideal for microbiological method, yielding the greatest amount of total folates. On the other hand, a modified method where deconjugation was conducted after successive treatments with amylase, protease and AC showed the best deconjugation efficiency and a slightly lower MA-derived content.

Meanwhile, different procedures exhibited varying discrimination to vitamers, resulting in varied recoveries for certain folates. However, other possible factors such as the type of sample and the MA response to AC eluent should be further investigated. Nevertheless, it is certain that appropriate pretreatments should be carefully selected and optimised according to the purpose of the study and the determination method.

With the commitment to food analysis, the validated AC-UPLC-FLR/PDA method was successfully applied for the identification of native 5-CH3-H4PteGlu1-8 in the lupin flour, faba bean flour and dry yeast, which underwent standard pretreaments omitting deconjugation. The UPLC results of the total folates showed good agreement with MA determinations. As the major species, the methylated folate pools were predominated by pentaglutamate in legume flours and heptaglutamate in dry yeast. In addition, based on

comparison of the results of deconjugated and undeconjugated samples, it was reasonable to conclude that other polyglutamylated species were also present in the samples, especially in legumes. This finding, therefore, raised the necessity to develop a universal method for all possible polyglutamylated derivatives when commercial standards were available. Also, the recoveries of other polyglutamates on AC should be carefully investigated in further studies.

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In document Approaches to folyl polyglutamate analysis (sivua 60-76)