Nutrition 26 (2010) 835–841
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Basic nutritional investigation
Supplementation with engineered Lactococcus lactis improves the folate status in deficient rats Jean Guy LeBlanc Ph.D. a, Wilbert Sybesma Ph.D. b, c, Marjo Starrenburg Ph.D. b, c, Fernando Sesma Ph.D. a, Willem M. de Vos Ph.D. b, Graciela Savoy de Giori Ph.D. a, d, *, Jeroen Hugenholtz Ph.D. b, c a
´n, Argentina Centro de Referencia para Lactobacillos (CERELA-CONICET), Tucuma Wageningen Centre for Food Sciences, Wageningen, The Netherlands NIZO Food Research, Ede, The Netherlands d ´n, Tucuma ´n, Argentina Universidad Nacional de Tucuma b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 May 2009 Accepted 26 June 2009
Objective: The aim of this study was to establish the bioavailability of different folates produced by engineered Lactococcus lactis strains using a rodent depletion–repletion bioassay. Methods: Rats were fed a folate-deficient diet, which produces a reversible subclinical folate deficiency, supplemented with different L. lactis cultures that were added as the only source of folate. Three bacterial strains that overexpressed the folC, folKE, or folC þ KE genes were used. These strains produce folates with different poly glutamyl tail lengths. The growth response of the rats and the concentration of folates in different organs and blood samples were monitored. Results: The folate produced by the engineered strains was able to compensate the folate depletion in the diet and showed similar bioavailability compared with commercial folic acid that is normally used for food fortification. Folate concentrations in organ and blood samples increased significantly in animals that received the folate-producing strains compared with those that did not receive bacterial supplementation. Hematologic studies also showed that administration of the L. lactis strains was able to revert a partial megaloblastic anemia caused by folate deficiency. No significant differences were observed in the bioavailability of folates containing different glutamyl tail lengths. Conclusion: To our knowledge, this is the first study that demonstrated that folates produced by engineered lactic acid bacteria represent a bioavailable source of this essential vitamin. Ó 2010 Elsevier Inc. All rights reserved.
Keywords: Folate Lactic acid bacteria Genetically modified microorganisms
Introduction Folates are water-soluble vitamins that play a crucial role as cofactors in human one-carbon transfer reactions. They are involved in the metabolism of nucleic acids and amino acids, which explains why they are essential for the growth and development of all cells. It has been shown that folate shortage may increase the risks of developing pathologies such as anemias, neural tube defects, cardiovascular diseases, and some forms of cancer [1–7]. Although folates are omnipresent in a normal human diet, folate deficiencies still occur frequently This work was supported by Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (ANPCyT) and the European Commission through contract QLK1-CT-2000-01376 (Nutracells). * Corresponding author. Tel.: þ54-381-431-0465; fax: þ54-381-400-5600. E-mail address:
[email protected] (G. S. de Giori). 0899-9007/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.06.023
even in well-developed countries [8,9], reason for which many countries now possess mandatory folate-fortification programs. Besides the total amount of folate that is consumed, the type of folate can also influence its effective uptake. Many food products are currently enriched with folic acid (pteroylglutamic acid), which is the synthetic form of folate that does not exist in nature. Too high of an intake of folic acid may mask the diagnosis of a vitamin B12 deficiency and possibly lead to irreversible neurologic damages [10], but this is not expected to occur with the naturally occurring folates found in food [11]. A recent report also has suggested that folic acid supplementation during pregnancy is associated with increased risks of respiratory infections in newborn children [12]. These researchers acknowledge that folic acid may act differently than natural occurring folates and that regular intake of folic acid supplements could result in circulating unmetabolized folic acid, which in turn may have unknown effects on immune cells by epigenetic mechanisms.
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It has been reported that the relative bioavailability of dietary folates is estimated to be only 50% compared with the synthetic folic acid [13]. Bioavailability is defined as the proportion of a nutrient ingested that becomes available to the body for metabolic processes or storage. The bioavailability of dietary folate may be hampered by the presence of cellular matrices in foods or the polyglutamate chain to which most of the natural folate is attached [14]. This polyglutamate chain must be removed in mammals, except for the proximal glutamate moiety, by glutamate carboxypeptidase II that is present in the brush border of the small intestine. Rats do not have intestinal brushborder folate conjugase; it is most likely that intracellular folate conjugase catalyzes the peptidase reaction in these animals [15]. Subsequently, folate can be absorbed and transported as mono glutamyl folate into the portal vein. In previous decades, several attempts have been made to assess the bioavailability of mono glutamyl folate compared with poly glutamyl folate. The available data suggest that the polyglutamate form is 60% to 80% bioavailable compared with the monoglutamate form [16,17]. Lactic acid bacteria, such as the industrial starter bacteria Lactococcus lactis and Streptococcus thermophilus, have the ability to synthesize folate [18,19]. For this reason, some fermented dairy products, including yogurt, are reported to contain even higher amounts of folate than non-fermented milk. It has also been shown that metabolic engineering can be used to increase folate levels in L. lactis [20,21] and Lactobacillus gasseri [22]. Our group has generated L. lactis strains that produce folates intracellularly with short or long poly glutamyl tail lengths (average number of glutamyl residues from three to eight), which generates an increased retention of folate in the cells [23]; however, the bioavailability of these folates has never been established. The present study describes the effects of feeding folatedepleted rats with genetically modified bacteria that overexpress specific folate biosynthetic genes with the aim to determine 1) whether folate-overproducing bacteria can be used as an alternative source of folate and 2) whether the folate glutamyl tail length affects its bioavailability.
Materials and methods Bacterial strains, media, and culture conditions Lactococcus lactis NZ9000 strains harboring the plasmids pNZ7010, pNZ7011, or pNZ7016 were used in this study [23]. These plasmids contain genes involved in the folate biosynthesis pathway of L. lactis that are considered essential for the modulation of the folate poly glutamyl tail length. Plasmid pNZ7016 contains the folC gene, encoding the bifunctional protein folate synthetase/poly glutamyl folate synthetase, under the control of the nisA promoter. Strains with this plasmid produce intracellular folates with an average of eight glutamyl residues [23]. Plasmid pNZ7010 contains the folKE genes under the control of the nisA promoter encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and guanosine triphosphate/ cyclohydrolase I, under the control of the nisA promoter and strains that possess this vector contain intracellular folates with an average of three glutamyl residues [23]. Plasmid pNZ7011 contains the folC and folKE genes under the control of the nisA promoter. The intracellular folate levels of strains with pNZ7011 are higher than in the wild-type strains and have folates with an average of 3.3 glutamyl residues [23]. Lactococcus lactis strains were grown at 30 C in M17 medium (Merck, Darmstadt, Germany) [24] supplemented with 0.5% glucose and 10 mg/mL of chloramphenicol. An overnight culture was diluted 1:100 in 5 L of the medium. Nisin induction was performed as described previously [20]. Several such batch fermentations were performed to obtain the desired amount of cells with intracellularly accumulated folate. Cells were harvested by centrifugation (6000 g, 30 min) and washed with 0.1 M sodium acetate containing 1% (w/v) ascorbic acid, pH 4.75. Subsequently, cells were freeze dried in dark during 48 h and were homogenously mixed with the folic acid–deficient diet (see Experimental Design).
Folate analysis Intracellular folate concentrations of the lyophilized cells were quantified using a microbiological assay and confirmed by high-performance liquid chromatography as described previously [19,25]. The latter technique was also used to determine the average poly glutamyl tail length. Experimental design The overall experimental protocol is summarized in Figure 1. Weanling male Wistar rats (mean standard deviation 60 3 g) were obtained from the closed colony of Universidad Nacional de Tucuma´n (Tucuma´n, Argentina). Rats were individually housed in wire-bottomed cages in a room with a 12-h light cycle at 22 2 C and were allowed free access to food and water throughout the study. A folic acid–deficient diet (FADD; MP Biomedicals, Irvine, CA, USA) containing 1% succinylsulfathiazole (MP Biomedicals) was used in this study. The composition of this diet is presented in detail in Table 1. Sixty-four rats were randomly selected for the study and were divided into two groups of 8 and 56 rats of equal mean weights. The first group of rats (n ¼ 8) was fed the FADD with 2 mg of folic acid per kilogram of diet (control group). The 56 remaining rats were fed the FADD for 30 d (depletion period). After the depletion period, eight rats were randomly selected and sacrificed. The 48 remaining rats were divided into six experimental groups, each containing eight rats (depleted–repleted group). These animals were fed with the same diet supplemented with different levels of folic acid (pteroylglutamic acid, SigmaAldrich, St. Louis, MO, USA; 125, 250, or 500 mg/kg of diet) or with engineered lactic acid bacteria that produce folates (250 mg of folates per kilogram of diet from L. lactis NZ9000 harboring pNZ7016 [folC], pNZ7011 [folC þ KE], or pNZ7010 [folKE]) during 28 d (repletion period). Animal live weight and food consumption were determined during the depletion and repletion periods. All animal protocols were approved by the animal protection committee of CERELA and followed the latest recommendations of the Federation of European Laboratory Animal Science Associations. All experiments complied with the current laws of Argentina. Blood and organ sample collection When required, animals were anesthetized with an intraperitoneal injection of 3.0 mL of ketamine (10% w/v)–xylazine (2% w/v; 40:60 v/v, Alfasan, Woerden, The Netherlands) per kilogram of animal weight and bled by cardiac puncture. Blood was transferred into tubes with or without anticoagulant (Heparin, Rivero, Buenos Aires, Argentina). Blood smears were prepared immediately from samples taken with anticoagulant and were stained with Giemsa (Biopur Quimica, Argentina). These same samples were used for hematological studies. Red blood cells, white blood cells, hemoglobin, hematocrit, white blood cell differential counts, mean corpuscular volume, mean corpuscular hemoglobin concentration, and mean corpuscular hemoglobin were determined according to the current guidelines of the Colegio Bioquı´mico de Tucuma´n (Tucuma´n, Argentina). For serum samples, blood without anticoagulant was allowed to clot; serum was separated by centrifugation (1500 g for 10 min) and diluted 1/10 with assay buffer (0.1 M K2HPO4/KH2PO4 buffer containing 0.3% ascorbic acid). For preparation of whole-blood samples for erythrocyte folate analysis, an aliquot of blood containing anticoagulant (100 mL) was diluted in 9 vol of distilled water (900 mL) and incubated for 30 min at 37 C to allow serum conjugase to convert folate polyglutamates released from the lysed erythrocytes to the assayable monoglutamate forms. These lysates were then diluted (500 mL in 4.5 mL) in the assay buffer. For plasma samples, remaining blood containing anticoagulant was separated by centrifugation (1500 g for 10 min) and diluted 1/10 with assay buffer. The diluted blood samples were further processed as described previously [26]. For organ samples, aliquots (0.5 g) of freshly excised organs (liver, spleen, and kidneys, which were weighed to determine organ weight ratios) were added to 9 vol (w/v) of assay buffer, homogenized for 1 min, and incubated at 37 C for 24 h to allow the endogenous conjugase to convert folate polyglutamates to the assayable monoglutamate forms. Samples were further processed as described previously [26]. All samples were analyzed for folate content by using a microbiological assay as described earlier. Statistical analysis All values are expressed as means standard deviations. Differences were evaluated using one-way analysis of variance followed by Tukey’s post hoc comparisons. Statistical analyses were performed with Minitab 14 (Minitab Inc., State College PA, USA), and differences were considered statistically significant at P 0.05.
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Fig. 1. Folate depletion–repletion experimental protocol. Letters in parenthesis indicate when B or O samples were taken. Italicized numbers represent micrograms of folic acid per kilogram of a folic acid–deficient diet or the different lactococcal strains (expressing the folC, folKE, or folC þ KE genes) that were added to the same diet (250 mg/kg of diet). B, blood; O, organs.
Results The hematologic studies showed symptoms that indicated that a partial megaloblastic anemia was present in folatedepleted animals (Fig. 2 and Table 2). In the depleted animals, erythrocyte (red blood cell) and white blood cell numbers were lower, as were hemoglobin and hematocrit concentrations, whereas mean corpuscular volume and mean corpuscular hemoglobin levels were higher compared with those observed in the non-depleted group (control group, Table 2). Furthermore, morphologic observations of blood smears showed that, Table 1 Composition of the folic acid deficient diet Ingredients
g/kg dry feed
Gelatin Dextrose Corn oil Hydrogenated vegetable oil Vitamin-free casein Salt mix Briggs* Vitamin fortification mixturey
80 455 20 185 200 50 10
* Salt mixture composition (percentage): calcium carbonate 16.67; calcium phosphate 47.3; copper sulphate 0.017; ferric citrate 0.333; magnesium sulphate 5.0; manganese sulphate 0.417; potassium chloride 11.67; potassium iodate 0.017; sodium chloride 6.67; sodium phosphate dibasic 11.67; zinc carbonate 0.217. y Vitamin amount per kilogram of diet: vitamin A acetate 19 800 IU; calciferol 2210 IU; a-tocopherol 121 IU; inositol 0.11 g; choline chloride 1.672 g; menadione 49.3 mg; biotin 0.5 mg; p-amino benzoic acid 0.11 g; ascorbic acid 0.99 g; niacin 98.1 mg; riboflavin 22 mg; pyridoxine HCl 22 mg; thiamine HCl 22 mg; calcium pantothenate 66 mg; vitamin B12 29.9 mg.
after the depletion period, red blood cells were slightly hypochromic and some neutrophil granulocytes showed multisegmented nuclei (Fig. 2b). At the end of the repletion period, the hematologic values returned to normal values (no statistical differences compared with those of the control group) as did the morphology of the blood cells in the animals that received the folates from the engineered L. lactis (expressing folC, folKE, or folC þ KE) or in animals given the same amount (250 mg/kg of diet) of commercial folic acid (Fig. 2d,e,f,c respectively). The mean folate concentrations in selected organ (liver, kidney, and spleen) and blood (erythrocytes, plasma, and serum) samples are shown in Figure 3. A significant decrease in folate concentrations in liver and kidney samples (Fig. 3a) was observed in the depleted animals compared with those that were not depleted (control group). The animals that received the folate-producing strains showed an increase in tissue folate concentrations compared with those observed in the depleted group; no significant differences were observed in these latter groups compared with the animals that received the same amount (250 mg/kg of diet) of commercial folic acid. However, there were no significant differences in folate levels of animals fed with the different L. lactis cells that produced folates with different poly glutamyl tail lengths. Furthermore, a linear relation between the concentration of folic acid added in the standard diet groups (that received 125, 250, and 500 mg of folic acid per kilogram of diet) was observed; the mean liver and kidney folate concentrations can be described by the equations y ¼ 0.0146x þ 8.62 (R2 ¼ 0.79) and y ¼ 0.0132x þ 3.64 (R2 ¼ 0.77), respectively, where y is the expected concentration of folate
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(micrograms) per gram of organ, and x is the concentration of folic acid (micrograms) per kilogram of diet. Based on these equations, the folate concentration in the diets with the lactococcal cells could be estimated, and the results demonstrated that the bioavailability of the folates produced by these strains was slightly higher than those of the animals fed the 250-mg folic acid/kg diet (data not shown). Mean folate concentrations in the spleen, serum, plasma, and erythrocytes of rats fed 125, 250, or 500 mg of folic acid per kilogram of diet were not significantly different within the same sample type (organ or blood samples). Therefore, corresponding regression equations were not calculated and no further interpretations could be made. In serum samples, a significant decrease of folate concentrations was observed in the depleted animals (Fig. 3b) compared with those in the control group. The animals that received the L. lactis strains harboring plasmids pNZ7016 (folC) and pNZ7011 (folC þ KE) showed a significant increase in folate concentrations compared with the depleted animals, as did those that were fed with 250 mg of folic acid per kilogram of diet. The folate concentration in erythrocytes did not vary significantly (Fig. 3b); the values of the non-depleted, depleted, and
depleted–repleted animals were not statistically different. Plasma folate concentrations decreased significantly (Fig. 3b) in the depleted animals compared those in the control group; these lower concentrations were maintained in the depleted–repleted animals that were fed the lactococcal strains or commercial folic acid during the repletion period. No significant differences were observed in the growth rates, final growth weights, and organ weight ratios of the rats fed the lactococcal strains (expressing folC, folKE, or folC þ KE), different commercial folic acid (125, 250, or 500 mg of folic acid/kg of diet), or the control group. Food consumption during the repletion period did not vary significantly between the different experimental groups that were given the FADD supplemented with the lactococcal strains or commercial folic acid (data not shown). Discussion The present study was conducted to assess the effects of administering engineered lactic acid bacteria, which overexpress specific folate-producing genes, conferring them the ability to produce folates with different poly glutamyl tail lengths, in
Fig. 2. Blood smears (magnification 1000) of rats fed (a) a folic acid–deficient diet supplemented with 2 mg of folic acid throughout the trial (control group), (b) a folic acid– deficient diet during 30 d (depleted), a folic acid–deficient diet during 30 d followed by 28 d with a folic acid–deficient diet containing (per kilogram): (c) 250 mg of folic acid or 250 mg of folate Lactococcus lactis containing (d) pNZ7016 (folC), (e) pNZ7010 (folKE), or (f) pNZ7011 (folC þ KE). Arrow points to a hypersegmented neutrophil that is characteristic of megaloblastic anemias.
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Table 2 Hematologic values of rats fed a balanced rodent food (control), a folic acid–deficient diet supplemented with 2 mg of folic acid/kg of diet throughout the trial (normal), a folic acid–deficient diet during 30 d (depleted), a folic acid–deficient diet during 30 d followed by 28 d of feeding with a folic acid–deficient diet containing (per kilogram of diet) 250 mg of folic acid or 250 mg of lactococcal folate (from Lactococcus lactis expressing folC, folKE, or folC þ KE)* Variable
Control
RBCs (106/mm3) Hemoglobin (g/dL) Hematocrit (%) MCV (fl) MCHC (%) MCH (pg) WBCs (103/mm3) Neutrophils (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) Basophils (%)
7.0a 14a 41a 58.7a 33.2 19.5a 14b 5 92 2 1.3 1
Depleted
Folic acid 250 mg
5.4b 11.7b 33b 62b 35 22b 3c 10 82 3 3 13
7.1a 14a 41a 58a 34 19.7a 6 9 87 2 1.0 0.6
Lactococcus lactis folC
0.7 1 4 0.7 0.7 0.6 2 2 3 1 0.5 1
0.1 0.7 1 1 3 1 1 3 5 2 1 0.6
0.6 1 4 1 1 0.8 2 4 5 1 0.8 0.5
7.2a 14.3a 42a 58a 34 20a 7.0c 10 85 3 1.5 0.9
folKE
0.5 0.5 2 2 1 1 1.5 5 4 2 0.9 0.8
7.4a 14a 43a 58a 33 19.3a 8c 9 80 3 6 2
folC þ KE
0.7 1 4 2 1 0.6 2 5 19 2 13 1
7.5a 14.5a 44a 59a,b 33 20a,b 11a 7 90 1.7 0.6 1.3
0.7 0.5 3 2 1 1 2 2 2 0.5 0.5 0.5
MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; RBCs, red blood cells (erythrocytes); WBCs, white blood cells * Values are means SDs. a,b,c Mean values within a row with unlike superscript letters were significantly different (P < 0.05).
folate-depleted rodents. The setup of the rat bioassay was based on previously described models [26] using a folate depletion– repletion diet that also included groups of animals that were fed with different concentrations of commercial folic acid during the repletion phase. In our model, succinylsulfathiazole was added to prevent the growth of potential folate-producing micro-organisms that are sometimes present in the indigenous microbiota of rodents [27]. A partial megaloblastic anemia was observed using this model in the rats that were deprived of all folate sources during 30 d. This anemia was reversible because the hematologic values and morphology of the blood cells returned to normal after the administration of the repletion diets. Animals that received folates from the engineered L. lactis strains (expressing folC, folKE, or folC þ KE) showed similar hematologic values as the animals given the same amount of commercial folic acid. These results clearly show that the bacterial strains used in this study produce folates that are able to revert a megaloblastic anemia caused by folate deficiency in rodents. Because no significant variations were observed between the groups of animals that received the different L. lactis strains, the poly glutamyl tail length did not seem to affect the bioavailability of the bacterially synthesized folates. Several studies have reported a wide range of folate concentrations found in rat tissues after performance of similar rat bioassays. In this study, the folate concentrations determined in various tissue and blood samples of the rats were analyzed in two independent laboratories (CERELA-CONICET in Argentina and Nizo Food Research in the Netherlands) and were shown to be almost identical for all the samples tested (data not shown). Overall, our values are consistent with what has been reported previously except for erythrocyte folate concentrations [26,28]. A significant decrease in folate concentrations in the liver, kidney and serum was observed in the folate-depleted animals compared with those that were not deprived of this essential vitamin. The animals that were repleted with the folateproducing strains showed an increase in tissue folate concentrations. As was the case for the hematologic values, there were no significant differences in folate levels of animals fed with the different L. lactis cells that produced folates with different poly glutamyl tail lengths, confirming that folate bioavailability does not seem to be affected by the amount of glutamyl residues present on the folate molecules.
The low folate levels found in the erythrocytes implies that the repletion phase might not be sufficient; increasing the duration or the concentrations of folate added to the diet might be necessary in future trials to circumvent this problem. Another point of interest is related to the absence in our study of an important response between the serum and plasma values determined in the depleted group and in the control group. It might be possible that the amount of folic acid given to this latter group of animals (2 mg of folic acid/kg of diet) is not sufficient for a maximum response in these samples or the repletion period might not be long enough. The absence of a linear response in plasma, serum, and erythrocytes may be due to the low concentrations of folate in these samples compared with folate concentrations in organs, in combination with the sampling method that was used in this study and in previous works. Recently, we determined that heating of samples with low folate concentrations partially degrades folates; this effect is not observed with high folate concentrations (Starrenburg, unpublished results). Therefore, for future rat bioassays we recommend omitting extensive sample preparation to adequately determine folate in blood samples. Furthermore and contrary to previous works [29], growth was not a good parameter to assess folate bioavailability in the present study. This is probably due to the commercial folatedeficient diet used in this study because it was previously demonstrated that differences in growth can be observed only using folate-free amino acid–based diets and not in casein-based diets [30]. A folate-free amino acid–based diet produces severe megaloblastic anemia and growth retardation. In this study, a casein-based diet was chosen to produce a reversible subclinical folate deficiency that is the most common manifestation of folate deprivation present in most societies. Post enzymatic conjugation with an external enzyme source (from human plasma, Sigma-Aldrich) in blood and organ samples showed that no residual poly glutamyl folates were present (data not shown), confirming that endogenous conjugase activity is present in these samples, as was previously reported [26]. In this rat feeding trial, the folate poly glutamyl tail length did not appear to affect its bioavailability. This is clearly different from what has been reported for humans [14,16,17]. The rat carboxypeptidase II enzyme that is required for transforming
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Fig. 3. Folate concentrations determined in organs (a) and blood (b) after folate depletion–repletion periods of rats fed a folate-deficient diet supplemented with 2 mg of FA throughout the trial (control), a 30-d depletion diet (depletion), or a 30 d depletion diet followed by a 28-d repletion diet containing 250 mg of lactococcal folate (folC, folKE, or folC þ KE overexpression) or 125, 250, or 500 mg of folic acid per kilogram of diet. Values are means SDs. Mean values within a sample type (tissue or blood sample) with unlike superscript letters were significantly different (P < 0.05). FA, folic acid.
poly glutamyl folate into mono glutamyl folate, which can subsequently be absorbed by the blood, obviously does not limit the folate absorption of poly glutamyl folates with different glutamyl tail lengths. Using the equations obtained from the linear response to different concentrations of folic acid in the liver and kidney samples, the absolute folate levels determined in the organs seem to indicate that more than the earlier determined 250 mg of folate from lyophilized cells was added to each kilogram of diet (data not shown). An explanation may be that the microbiological assay that was used to determine the folate concentrations in the different bacterial cells underestimates the natural folates compared with folic acid (pteroylglutamic acid) that is traditionally used as a standard in this assay. However, high-performance liquid chromatographic analysis of the different samples shows that similar amounts of the bacterial folates to those of the chemical folic acid were present in the diets. An alternative explanation would be that the bioavailability of the natural folates is higher than that of folic acid, but this is not supported by previous studies [13,28,31]. However, it is important to emphasize that this is the first study that describes the bioavailability of folates with differing poly glutamyl tail lengths produced by food-grade micro-organisms.
The ability of L. lactis to survive the hostile conditions of the human gastrointestinal tract has made it the model bacterium for the delivery of gene products in the gut [32]. The survival, physiology, and lysis of this lactic acid bacterium in the digestive tract have been extensively studied [33]. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. These properties suggest that L. lactis could be used as a vector to specifically deliver vitamins (or other compounds) produced intracellularly into the duodenum of monogastric animals. The outcome of this study revealed that L. lactis can be used as a delivery vehicle for folate just as previously shown for riboflavin using L. lactis [34,35] and Propionibacterium freudenreichii [36]. In contrast to mono glutamyl folate, poly glutamyl folates cannot be transported across the cell membrane. Hence, the release of intracellular poly glutamyl folate depends on the disruption of the cells during passage through the gastrointestinal tract. The clear responses of lactococcal cells added to the folate-deficient diet on the folate concentrations in organs and blood indicate that these cells lyse after consumption and that the bacterial folate becomes available for absorption in the gastrointestinal tract of the rat. Although the folate-producing capabilities of food-grade and potentially probiotic micro-organisms such as lactic acid bacteria [19], bifidobacteria [18,37–39], and propionibacteria [38,40] have been published for some time now, few studies have been performed to demonstrate that this production could have beneficial effects on the host. Recently, it was shown that the administration of folate-producing bifidobacteria could increase folate concentrations in the serum and liver [41]. It was also shown that the simultaneous administration of prebiotics (oligofructose) together with these folate-producing microorganisms enhanced their effectiveness on the folate status of the depleted rats [41]. These results indicate that the composition of the diets should be taken into account when evaluating folates, especially those that are synthesized by micro-organisms during food production, because its bioavailability could be affected by compounds normally present in foods. This study has provided the first animal trial with food containing living bacteria that were engineered to increase the intracellular accumulation of folate by modulation of the average poly glutamyl tail length. These results pave the way for analyzing the effect of these folate-overproducing lactic acid bacteria in human trials. These strains could be used in the development of novel fermented foods bio-enriched with ‘‘natural’’ folates, which could be used as an alternative to fortification with folic acid. Acknowledgments The authors thank Analı´a Rossi and Silvia Burke for their help with the care of the animals and sampling and Arno Wegkamp for analyzing the folate samples by high-performance liquid chromatography. References [1] MRC Vitamin Study Research Group. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet 1991;338:131–7. [2] Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 1995;274:1049–57. [3] Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med 1992;327:1832–5.
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