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Mal J Nutr 18(3): 383Folate - 391, Content 2012 and Availability in Malaysian Cooked

Folate Content and Availability in Malaysian Cooked Foods

Chew SC1, Khor GL2 & Loh SP1,3*

1 Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra , 43400 Serdang, Selangor, Malaysia 2 Department of Nutrition and Dietetics, Faculty of Medicine and Health, International Medical University, 57000 Kuala Lumpur, Malaysia 3 Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

ABSTRACT

Introduction: Data on folate availability of Malaysian cooked foods would be useful for estimation of dietary folate intake; however such information is scarce. Methods: A total of 53 samples of frequently consumed foods in Malaysia were selected from the Nutrient Composition of Malaysian Foods. Folate content

was determined using HPLC method hyphenated with a stainless steel C18 column and ultraviolet detector ( = 280 nm). The index of folate availability was defined as the proportion of folate identified as monoglutamyl derivatives from the total folate content. Results: Total folate content of different samples varied from 30-95 µg/100g fresh weight. Among rice-based dishes, the highest and the lowest total folate was in coconut milk rice () and rice (), respectively. In noodle dishes, fried rice noodle (kuey teow goreng) and noodle (mee kari) had the highest folate contents. The highest index of folate availability was in a flat rice noodle dish (kuey teow ) (12.13%), while the lowest was in a festival ( bakul) (0.13%). Folate content was found to be negatively related to its availability. Conclusion: This study determined folate content and folate availability in commonly consumed cooked foods in Malaysia. The uptake of folate from foods with high folate content may not be necessarily high as folate absorption also depends on the capacity of intestinal deconjugation and the presence of high fibre in the foods.

Keywords: Folate content, availability, HPLC

INTRODUCTION 2012). The only difference between folic acid and a folate is that folate is present Folates are mainly found in vegetables, naturally in the food whereas folic acid is in fruits, legumes and cereal products. It is a B the form of vitamin applied mainly during vitamin which constitutes various mono- fortification. Natural food folates are highly and pteroylpolyglutamates (two to seven sensitive to heat, hence, cooking can result glutamyl residues) of pteroic acid and has in destruction of 50-95% of the folate nutritional benefits similar to folic acid contents of food. besides its chemical structures (Gregory,

* Correspondence author: Loh Su Peng; Email: [email protected] Chew SC, Khor GL & Loh SP

Folate plays a crucial function in is defined as the fraction of ingested folate synthesising components of DNA, RNA and that is absorbed and can be used for proteins. Besides, it is essential for metabolic processes (Melse-Boonstra, hematopoiesis as well (Klee, 2000). Hence, Verhoef & West 2004). The bioavailability of deficiency in this vitamin causes the folate from the diet is less than 100% due to occurrence of megaloblastic anaemia and several factors including the species of folate, neural tube defects (Gujska & Kuncewicz, linkages at molecular level, amount of folate 2005). Further, the elevation of plasma and folic acid consumed, matrix, effect of homocysteine could be due to folate modifiers, nutrient status, genetic factors, deficiency (Gujska & Kuncewicz, 2005). host-related factors, and mathematical Worldwide, attention and programmes have interactions between the various factors been initiated to encourage a higher intake (Melse-Boonstra et al., 2004). Currently data of folate. However, in many countries on folate content and availability of cooked including Malaysia, the intake of folate has foods consumed by Malaysians are scarce. been found to be below the recommended This data would be seen as necessary to help intake (Khor et al., 2006, Chew, Khor & Loh, in evaluating the adequacy of folate intake 2011). especially among Malaysians. Therefore, the It has been reported that analysis of objective of this study was to determine folate folate in foods is not easy, due to its multiple content and availability of commonly forms, lower stability, presence at low consumed foods among Malaysians, using concentrations in biological systems, and the HPLC method. complex extraction and detection techniques (Devi et al., 2008). Generally, the most METHODS common and widely used method for folate analysis is microbiological assay. However, Chemicals and reagents the disadvantage of using this method is it Sodium ascorbate, pepsin, sodium chloride, only provides a total figure for all the tetrabutyl ammonium phosphate, dithio- derivatives of folic acid and does not erythritol, sodium acetate, hydrochloric acid, distinguish between individual forms of mannitol, calcium chloride, HEPES, zinc , folate (Johnsson Jagerstad & Frolich, 2007; acetate, 5-methyltetrahydrooflate (disodium et al et al Rychlik ., 2007; Poo-Prieto ., 2006). salt), folic acid, dimethyl glutaric acid and Another approach is using affinity bis (2-hydroxyethyl) imino-tris (hydroxy- chromatography in combination with a methyl) methane (Bis-Tris) were obtained high-performance liquid chromatography from Sigma Chemical (St. Louis, MO, USA). (HPLC) diode array ultraviolet (UV) Acetonitrile and water were of HPLC grade detection system. This has been used lately (Merck, Darmstadt, Germany). for folate determination to distinguish the single vitamers. Compared to microbiological Preparation of jejunal brush border assay, HPLC is a simple and more rapid membrane from pig intestine procedure in determining folate content. The downside of using HPLC is the need to Pig intestine was chosen as the source of identify all the known forms of folates in pteroylpolyglutamate hydrolase due to the order to be able to quantify total folates in a similarities between the enzymes present in given sample, where some forms are hard to human and pig intestine (Gregory et al., find or costly (LeBlanc et al., 2007). 1987). The mucosa was scraped and the Knowledge about folate bioavailability brush border membrane vesicles were from food is essential for the estimation of prepared according to Selhub & Rosenberg dietary requirements. Folate bioavailability (1981). Firstly, the mucosal scrapings were Folate Content and Availability in Malaysian Cooked Foods homogenised for 30 s at 0oC in 15 volumes pH 7.8 (Seyoum & Selhub, 1993). After (w/v) of 0.05 M mannitol. Then, the cooling in an ice bath, the extract was further homogenate was centrifuged for 10 min at centrifuged (40000 x g, 20 min, 4oC) before 10000 x g. The pellet underwent a second storing the supernatant at -80oC. The folate homogenisation for 1 min by using fresh 15 content for each food was determined by the ml mannitol solution before being combined HPLC method according to Seyoum & with the first supernatant. The whole mixture Selhub (1993). For the experimental group, was then filtered. Solid calcium chloride was it first underwent digestion with acid/peptic added to the filtrate to make a final mixture. In this step, the homogenate was concentration of 10 mM before being acidified to pH 2 with 1 mol/L HCL and centrifuged again at 4000 x g for 10 min and mixed thoroughly with pepsin (0.5 g/100g 40000 x g at 15 min. The pellet was then re- food). Then, it was incubated for 2 h at 37oC suspended in 100mM mannitol containing in a shaking water bath before adjusting the 10 mM potassium/HEPES at pH 7.4. Finally, mixture to 6.5 with 1mol/ L NaOH. The the homogenate was centrifuged again mixture was then boiled at 120oC for 30 min (40000 x g, 15 min) and the pellet fraction in equal volume of extraction buffer (20 g/L was re-suspended in 2 ml mannitol sodium ascorbate and 100 mmol/L Bis-Tris potassium/HEPES solution, divided into buffer, pH 7.8) in order to extract the folates. aliquots and stored at -70oC. The extract was centrifuged at 40000 x g for 20 min at 4oC after cooling in an ice bath and Sample preparation the supernatant was then divided into two A total of 53 samples of frequently consumed equal portions in order to determine cooked foods in Malaysia were selected susceptibility of folates to pteroylpoly- based on the foods analysed in the Nutrient glutamate hydrolase action before and after Composition of Malaysian Foods (Tee et al., purification by high-performance liquid 1997). The foods chosen were rice-based, chromatography. noodles, local (kuih-muih), and breads. All the samples were purchased from Incubation before purification different types of stalls in Serdang, Selangor One portion of the supernatant was as described in Chew, Loh & Khor (2012). incubated (37oC, 4h) with pig intestine (5 mg The foods were ground and homogenised protein), 33 mmol/L dimethylglutarate before analysis. Preparation was carried out buffer (pH 6.5), 0.25 mmol/L sodium under subdued light. Due to the physical chloride, 0.1 mmol/L zinc acetate and 2.5 characteristic of folate which is easily g/L sodium acetate solution in a shaking destroyed by heat, air and light during food water bath. The mixtures were determined preparation, the foods were analysed as by HPLC after the incubation as outlined by ready for consumption to determine the Seyoum & Selhub (1993). The absorbance of actual folate contents. The grounded food eluted folates was monitored at 280 nm using was divided into two equal portions; the a diode array detector. control group in which the folate was extracted under protective conditions and Incubation after purification analysed as such without treatment and the Another portion of the supernatant was experimental group where the extraction and subjected to purification on affinity column treatment was performed. For control group, before incubating with the brush border the foods was extracted by autoclaving for membrane preparation as described above. 30 min at 120oC in 10 vol 20g/L sodium After purification by affinity chromatography, ascorbate and 100 mmol/L Bis-Tris buffer, folate monoglutamates were measured Chew SC, Khor GL & Loh SP using HPLC method with diode array RESULTS AND DISCUSSION detection. All analyses were performed under subdued light. This procedure was Folate content quantification using HPLC used to determine the most abundant folate method forms naturally present in foods including Total folate content of different food samples 5-methyltetrahydrofolate. The calibration in this study varied from 30-95 µg/100g fresh curves were constructed by plotting peak weight (Table 1). Among the samples, area against concentrations. canai with egg (roti telur) had the highest amount of total folate, followed by fried HPLC condition kueyteow (kuey teow goreng) and The HPLC system (Hewlett Packard HPLC (popiah). Among the rice group, coconut milk Series 1100) was equipped with a stainless rice (nasi lemak) had the highest level of folate. Besides fried kuey teow (kuey teow goreng), steel column C18, 250 mm x 4.6 mm I.D (Hewlett Packard, USA). The chromato- curry noodle (mee kari) had the second graphic conditions were as follows: column highest folate level among the other noodles. temperature, 23oC; autosampler temperature, The highest total folate content in the kuih- 8oC; flow rate, 1ml/min; volume injected, 20 muih group was unleavened burnt sugar µL, and untraviolet detection (UV), 280 nm. (apam gula hangus), followed by popiah The mobile phase consisted of variable (popiah). For roti (bread) group, mixtures of 5mmol/L tetrabutyl ammonium showed the second highest folate level after phosphate, 25 mmol/L NaCI, 5 mmol/L roti canai with egg (roti telur). Among the dithioerythritol, and 10% acetonitrile. The foods studied, the lowest folate levels were index of folate availability was assessed by found in steamed tapioca parcels (lepatubi) comparing the concentration of the in the kuih-muih group. Eight foods had monoglutamylfolate in the experimental folate levels in the range of 50-100 µg/100g group to the folate concentration in the fresh weight; they were coconut milk rice control group as follows: (nasi lemak), fried kuey teow (kuey teow goreng), curry noodle (mee kari), and popiah, M1 Folate availability index = x 100 Chinese (yau cha kuai), unleavened T1 burnt sugar (apam gula hangus), roti canai with where M1 is the monoglutamylfolate egg (roti telur), and roti canai. The remaining concentration after treatment and T1 is the had lower concentrations with a range of total folate concentration in the control 30-50 µg/100g fresh weight. Some of the group. foods studied showed high coefficient variations for the total folate using HPLC Data analysis method. The variability observed in this study could be due to several factors. First, All data are reported as mean ± SD. The the ingredients used for the preparation of statistical analyses were carried out by using certain foods might differ from one location SPSS 17. The differences in the release of to another. Our study homogenised and monoglutamylfolate before and after the pooled samples from nine stalls obtained purification were compared by using paired from different locations around Serdang, sample T-test. The value was significant at Selangor. This could contribute in part to p < 0.05 level. Besides, the mean differences some of the variability found in this study. between the folate content with availability Additionally, the HPLC method has been was compared using independent sample shown to be lacking in stability during T-test and their relationships were extraction (Koontz et al.,2005) and this might computed using the Pearson correlation test. have an effect on the results obtained. Folate Content and Availability in Malaysian Cooked Foods 37.86 4.92 13.0 ) 52.02 3.92 7.5 ) 39.21 6.26 16.0 ) 33.19 1.98 6.0 ) 32.5 3.67 11.3 ) 33.11 0.64 1.9 ) 31.68 0.9 2.8 ) 34.6 2.46 7.1 Kuih bom ) 33.97 2.18 6.4 ) 34.47 0.55 1.6 ) 34.34 1.47 4.3 ) 50.98 3.98 7.8 ) 34.72 2.07 6.0 ) 32.11 1.22 3.8 ) 33.66 2.43 7.2 ) 33.91 1.64 4.8 ) 35.39 1.46 4.1 Apam gulahangus Lepat ubi Lepat pisang Rempenyek ) 34.41 1.1 3.2 Pulut panggang ) 43.37 1.2 2.8 )) 32.62 2.59 7.9 49.15 7.51 15.3 ) 44.26 4.72 10.7 Kuih ketayap ) 45.16 2.25) 5.0 37.73 3.62 9.6 ) 38.2 3.14 8.2 Kuih bakul Yauchakuai Keropok ubi ) 32.13 1.06 3.3 ) 38.87 3.75 9.6 Pau goreng Kuih serimuka Tumpi ) 33.99 2.49 7.3 Cekodok pisang Kuih bawang Cucur udang )Roti 62.64 0.95 1.5 g/100g fresh weight) Kuah dhal µ Keropok Kuih tiram Kuih kwaci Kuih lapis Karipap Donut muruku popiah Bahulu ( Banana ( Prawn fritter ( ( Currypuff ( Fried lekor ( Cassava crackers( New year’s cake ( ( Sweet potato and sesame balls ( Kuihkaswi ( Coconut filled crepe ( Black koci (Kuihkocipuluthitam) ( Draw pie cake ( Kuih tiram ( Steamed banana parcels ( Steamed tapioca parcels ( Muruku ( Fried dumpling( Fried banana( Popiah ( Grilled glutinous rice ( Savoury cracker ( Lace ( Fried pancake ( Chinese crullers ( SampleKuih-muih ( Unleavened burnt sugar ( Mean SD % CV Dhall gravy ( 94.7136.45 4.7 7.28 5.0 20 41.9536.35 8.8939.11 21.2 0.17 3.61 0.5 9.2 ) 42.83 4.75 11.1 ) 32.35 0.35 1.1 ) 94.6 8.78 9.3 ) 37.76 7.66 20.3 ) 40.72 9.47 23.3 ) 61.77 5.79 9.4 telur) ) 48.47 6.96 14.4 ) 36.83 4.71 12.8 ) 38.77 7.12 18.4 ) 34.18 1.66 4.9 ) 53.07 2.34 4.4 ) 36.95 6.44 17.4 ) 33.54 2.1 6.3 Kueyteow bandung ) 40.1 4.48 11.2 Meehoon bandung ) 55.09 5.18 9.4 Nasi lemak Meebandung Kueyteow goreng Meehoon goreng Mee kari Mee sup Nasi ayam ) 42.84 1.51 3.5 Nasi briyani i) 36.29 0.04 0.1 ) 48.53 4.06 8.4 roti canai Nasi minyak g/100g fresh weight) µ Folate content in foods capati g/100g fresh weight) thosa g/100g fresh weight) µ µ Table 1. Coconut milk rice ( Ghee rice ( Bandung meehoon ( SampleRice ( Chicken rice ( Briyani rice( Fied rice ( Kerabu rice ( Noodle ( Bandung kueyteow ( Fried kueyteow ( MeanBandung noodle ( Fried noodle ( SDCurry noodle ( Noodle ( % CV Fried meehoon ( Rojak ( Roti( Chocolate Tosai ( cake Capati ( Roti canai ( Roti canai with egg (Roti Chew SC, Khor GL & Loh SP

Based on our previous study on the folate availability although the strength of determination of folate content using relationship was weak (P = -0.336, p< 0.05). microbiological assay, the folate content in This may be due to an altered digestion raw rice was 16 µg/100 g dry weight, process resulting from either the high (wheat or rice) was 2 µg/100 g dry weight, quantity of foodstuff (e.g., exceeding the dry noodle (3 µg/100 g dry weight), rice capacity of intestinal deconjugation) or the noodle (meehoon) (11 µg/100 g dry weight), presence of high fibre in the food. atta for making capati (8 µg/100 g dry The combination of affinity chromato- weight), chickpea (10 µg/100 g dry weight), graphy using the immobilised folate binding and yellow dhal (4 µg/100 g dry weight) protein (FBP) in this study had provided for (Chew et al., 2012. Basically, most HPLC- additional benefit in terms of its function in measured folate contents were lower than relation to purification and concentration of values determined by the microbiological foods to achieve the final analysis. This is assay. It has been suggested that non-folate also supported by the study of Kariluoto, compounds could influence bacterial Vahteristo & Piironen (2001). This method growth causing a higher folate content when produced a very specific and efficient detected by microbiological assay (Konings purification. Bagley & Selhub (2000) found et al., 2001). Hence, this explains the large that folates are efficiently purified by affinity variation in the results obtained when column. As a result, non-folate compounds comparing these two methods. This is in such as reducing agents, added to the agreement with the study by Pfeiffer, Rogers extraction buffer to protect the labile forms & Gregory (1997) where the microbiological of folate, will be washed off in the affinity assay showed a slightly higher folate content column before eluting the folates into the in bread and spaghetti samples compared analytical column. Moreover, it enables the to HPLC methods. food extract to be concentrated ten-fold or more, hence, small endogenous folate Folate availability index concentrations present in foods can be 5-methyltetrahydrofolate may be produced determined with sufficient sensitivity using during the preparation or processing of UV detection (Pfeiffer, Rogers & Gregory, foods (Konings et al., 2001). The results for 1997). The addition of piperazine to the the experimental group which were digested affinity column helps to neutralise the acidic with pteroylpolyglutamate hydrolase effluent of the column. contained in the brush border membrane In conclusion, this study provides from the pig intestine is presented in Table information about folate content and folate 2. Subsequent to the purification steps, a availability in commonly consumed cooked more obvious release of monoglutamyl folate foods in Malaysia. Most of the folate content was seen compared to before purification. was found to be negatively related to its The highest folate availability index was in availability. This could be caused by kuey teow bandung (12.13%) whereas the alteration in the uptake of folates due to the lowest was found in the traditional cake reduction in the folate carriers or high fibre (kuih bakul) (0.13%). content. The data obtained are just Based on the t-test between folate content preliminary results and hopefully in the and availability, the results showed future, further studies could be carried out significant differences (p < 0.05) whereas to generate data on folate content and its based on the Pearson correlation test, folate availability from commonly consumed content was found to be inversely related to foods. Folate Content and Availability in Malaysian Cooked Foods 4.33 6.11 0.027 1.43 3.19 0.02 Before Purified p-value purificationfraction (%) (%) ) 1.81 2.12 0.026 ) 4.76 6.02 0.03 ) 3.76 4.44 0.03 ) 2.73 6.98 0.03 ) 1.97 2.73 0.03 ) 2.67 3.16 0.029 Kuih bom ) 2.15 2.9 0.029 ) 7.08 8.3 0.029 ) 3.01 5.71 0.029 ) 2.19 2.32 0.028 ) 1.56 2 0.02 ) 2.51 7.05 0.03 ) 3.03 7.12 0.029 ) 3.01 8.94 0.029 ) 3.9 4.29 0.028 Lepat ubi Lepat pisang Rempenyek ) 2.9 10.32 0.029 Pulut panggang ) 1.98 3.25 0.023 ) ) 2.29 11.08 0.024 ) 3.06 3.9 0.03 ) 2.93 3.86 0.021 Kuih ketayap ) 3.26 9.06 0.023 ) 2.61 3.72 0.027 ) ) 3.84 4.82 0.027 ) 1.58 12.9 0.03 Kuih bakul Keropok ubi Yauchakuai ) 2.47 3.88 0.026 Pau goreng Kuih serimuka ) 5.6 12.14 0.029 Cekodok pisang Tumpi ) 1.63 3.3 0.014 Kuih bawang Cucur udang Pisang goreng g/100g fresh weight) Kuah dhal µ Kuih lapis Keropok lekor Kuih kwaci Donut Karipap Kuih tiram muruku Bahulu (popiah Apam gulahangus Kuih koci pulut hitam ample New year’s cake ( Onion cracker( Sweet potato and sesame balls ( Grilled glutinous rice ( Savoury peanut cracker ( Lace pancake ( Fried pancake( Fried lekor ( Cassava crackers( Dhall gravy ( Kuihkaswi ( Fried banana( Popiah Chinese crullers ( Doughnut ( Currypuff ( Coconut filled crepe ( Black glutinous rice koci ( Layer cake ( Draw pie cake ( Kuihtiram ( Steamed banana parcels ( Steamed tapioca parcels ( Muruku ( Fried dumpling( Unleavened burnt sugar ( Bahulu ( Banana fritter ( Prawn fritter ( S Kuih-muih ( 7.42 0.025 6.25 0.026 3.48 7.07 0.028 2.04 6.07 0.028 Before Purified p-value purification (%)fraction (%) ) 2.58 14.71 0.024 ) 2.99 4.57 0.03 ) 2.13 3.66 0.018 ) 3.03 3.7 0.027 ) 3.08 3.54 0.025 ) 1.69 2.61 0.015 ) 1.19 1.83 0.014 ) 2.22 2.83 0.021 ) 3.73 4.41 0.026 )) 2.46 2.53 3.66 0.029 3.82 0.028 ) 1.05 1.26 0.019 ) 8.55 14.27 0.029 ) 7.33 10.15 0.027 ) 3.19 8.2 0.026 Kueyteow bandung Roti telur Meehoon bandung ) 1.85 4.22 0.018 Nasi lemak Mee bandung Kueyteow goreng Meehoon goreng Mee kari Mee sup Mee goreng ) 3.09 4.22 0.024 Nasi ayam Nasi briyani Nasi kerabu ) 2.84 6.14 0.028 ) 1.53 4.1 0.021 roti canai Nasi minyak g/100g fresh weight) Nasi goreng Folate availability index µ capati rojak thosai g/100g fresh weight) g/100g fresh weight) µ µ ample Table 2. S Bandung kueyteow ( Noodle soup ( Bandung meehoon ( Rice ( Chicken rice ( Briyani rice ( Fied rice ( Kerabu rice ( Coconut milk rice ( Ghee rice ( Noodle ( Fried kueyteow ( Bandung noodle ( Fried noodle ( Curry noodle ( Fried meehoon ( Rojak ( Roti( Butter cakeCapati ( Chocolate cake Banana cakeRoti canai ( Roti canai with egg ( 5.3 3.4 Chocolate swiss roll Tosai ( Chew SC, Khor GL & Loh SP

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