Nils-Gunnar Carlsson

Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli.

Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P(5), D/L-Ins(2,3,4,5)P(4), D/L-Ins(2,4,5)P(3) or D/L-Ins(1,2,4)P(3), D/L-Ins(1,2)P(2) or Ins(2, 5)P(2) or D/L-Ins(4,5)P(2) to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D-Ins(1, 2,3,5,6)P(5) isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F(1) and F(2) from wheat bran and the myo-inositol phosphates produced by fraction F(2). Biochim. Biophys. Acta 302, 326-328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P(5), D-Ins(2,3,4,5)P(4), D-Ins(2,4,5)P(3), Ins(2,5)P(2) to finally Ins(2)P (notation 6/1/3/4/5).

The potential of bifidobacteria as a source of natural folate

Aims:  To screen 19 strains of bifidobacteria for main folate forms composition in synthetic folate‐free and complex folate‐containing media.

Phytate degradation by human gut isolated Bifidobacterium pseudocatenulatum ATCC27919 and its probiotic potential

The growing awareness of the relationship between diet and health has led to an increasing demand for food products that support health above and beyond providing basic nutrition. Probiotics are live organisms present in foods, which yield health benefits related to their interactions with the gastrointestinal tract. Phytases are a subgroup of phosphatases that catalyse the desphosphorylation of phytate, which reduces its negative impact on mineral bioavailability, and generates lower inositol phosphates. The aims of this investigation were to (i) study the ability of the probiotic candidate Bifidobacterium pseudocatenulatum to degrade phytate in synthetic medium, to (ii) identify the lower inositol phosphates generated, to (iii) study its survival under conditions mimicking gastrointestinal passage and finally to (iv) assess adhesion of the bacteria to Caco-2 cells. The first steps of InsP(6) degradation by B. pseudocatenulatum phytate-degrading enzyme/s were preferentially initiated at the DL-6-position and 5-position of the myo-inositol ring. It suggests that the main InsP(6) degradation pathway by B. pseudocatenulatum by sequential removal of phosphate groups was D/L-Ins(1,2,3,4,5)P(5) or D/L-Ins(1,2,3,4,6)P(5); D/L-Ins(1,2,3,4)P(4); to finally Ins(1,2,3)P(3) and D/L-Ins(1,2,4)P(3)/D/L-Ins(1,3,4)P(3). This human strain also showed a notable tolerance to bile as well as a selective adhesion capacity (adhesion to control surfaces was zero), to human intestinal Caco-2 cells comparable to the commercial probiotic B. lactis. The phytate-degrading activity constitutes a novel metabolic trait which could contribute to the improvement of mineral absorption in the intestine as a nutritional probiotic feature with potential trophic effect in human gut.

Quantification of total fatty acids in microalgae: comparison of extraction and transesterification methods.

Determination of microalgaes’ fatty acid content is often done with chloroform and methanol according to the Bligh and Dyer extraction, though faster methods exist. A number of comparisons between the Bligh and Dyer and faster methods have resulted in contradicting data, possibly due to differences in algae used and the different versions of the Bligh and Dyer method applied. Here, various forms of direct-transesterification (D-TE) and two-step transesterification (2-TE), including three versions developed in our lab, are compared with the original Bligh and Dyer (Can J Biochem Physiol 37: 911–917, 1959) extraction and two modifications thereof (Lee et al. J AOAC Int 79:487–492, 1996, and our own acidified version) on microalgae with different cell walls: Isochrysis galbana, Nannochloropsis oculata, and Phaeodactylum tricornutum. In total, fatty acid extracts from 11 methods were separated and quantified by gas chromatography with mass spectrometry. Results show that, for N. oculata and P. tricornutum, methods based on chloroform–methanol underestimated the fatty acid content compared with the 2-TE and D-TE methods, which gave similar results. Moreover, D-TE methods are faster than chloroform–methanol methods and use chemicals that are less toxic. Of the D-TE methods, the ones using hydrochloric acid and sulfuric acid recovered the most fatty acids, while boron trifluoride recovered slightly less. The main qualitative difference between the fatty acids recovered was that the chloroform–methanol methods recovered less saturated fatty acids in P. tricornutum.

Pathway of phytate dephosphorylation by beta-propeller phytases of different origins.

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by the beta-propeller phytase of Shewanella oneidensis was established, which was then compared with that of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and B. amyloliquefaciens 45 beta-propeller phytases. The data demonstrate that all of these beta-propeller phytases dephosphorylate myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via d-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3. Thus, the beta-propeller phytases prefer the hydrolysis of every second phosphate over that of adjacent ones. This finding does not support previous phytate degradation models proposed by J. Kerovuo, J. Rouvinen, and F. Hatzack (2000. Biochem. J. 352: 623-628) and R. Greiner, A. Farouk, M. Larsson Alminger, and N.G. Carlsson (2002. Can. J. Microbiol. 48: 986-994), but seems to fit with the structural model given by S. Shin, N.C. Ha, B.C. Oh, T.K. Oh, and B.H. Oh (2001. Structure, 9: 851-858).

A new approach to measuring vitamin D in human adipose tissue using time-of-flight secondary ion mass spectrometry: A pilot study

Circulating concentrations of vitamin D, 25(OH)D, and 1,25(OH)2D are lower in obese than lean individuals, but little is known about the adipose tissue content of these molecules. The aim of this study was to explore the possibility to use time-of-flight secondary ion mass spectrometry (TOF-SIMS) to measure vitamin D and its metabolites in fat tissue in obese and lean subjects. Abdominal subcutaneous adipose tissue (SAT) biopsies were obtained from three lean and three obese women, and paired biopsies SAT and visceral adipose tissue (VAT) were obtained from three obese subjects during gastric bypass surgery. TOF-SIMS was used to measure vitamin D3, 25(OH)D3, and 1,25(OH)2D3 in adipose tissue. We found that vitamin D3, 25(OH)D3, and 1,25(OH)2D3 in adipose tissue can be measured with TOF-SIMS. In adipose tissue, vitamin D3 and its metabolites were located in adipocyte lipid droplets. The content of vitamin D3 (P=0.006) and 25(OH)D3 (P=0.018) were lower in SAT in obese compared with lean women. TOF-SIMS has the potential to semi-quantitatively measure vitamin D metabolites in adipose tissue, and offers a possibility to compare vitamin D levels in different depots and groups of individuals. It also gives the opportunity to explore the localization of vitamin D metabolites at a cellular level.

Degradation of phytate by Pichia kudriavzevii TY13 and Hanseniaspora guilliermondii TY14 in Tanzanian togwa.

The fermented cereal-based gruel togwa is used as weaning food for children in Tanzania. Togwa is rich in minerals but these are often not available for uptake in the human intestine due to natural inhibitors, such as phytate (IP(6)). The yeasts Pichia kudriavzevii TY13, Hanseniaspora guilliermondii TY14 and TY20, isolated from Tanzanian togwa, and selected for high phytase activity in complex yeast medium YPD, were now studied regarding their ability to degrade IP(6) in maize-based model togwa. A modified constitutively high-phytase producing Saccharomyces cerevisiae BY80 and commercial Aspergillus ficuum phytase were included for comparison. In addition, a strain of Lactobacillus plantarum was included in the model-togwa set-up. All yeasts in the study grew and reached final cell density 1.5-2 log units higher than the start value. S. cerevisiae BY80 degraded 85% of the IP(6) in 48 h; the same degradation level as with A. ficuum phytase (89%). Of the togwa-isolated yeasts, P. kudriavzevii TY13 and H. guilliermondii TY14 showed strong phytate degradation in the model-togwa; 95% or more of the initial IP(6) was degraded after 48 h. This corresponds to a remaining level of 0.4 and 0.3μmol IP(6)/g dw. Co-inoculation with L. plantarum did not increase IP(6) degradation. Moreover, fermentation with P. kudriavzevii TY13 yielded a successive increase in inorganic phosphate (P(i)), from 0.7 to 5.4 mM, suggesting a phytase production in TY13 which is fairly insensitive to P(i) repression. The study shows that phytate in a model togwa is available for yeast phytase enzymes, and addresses the importance of strain selection for effectively degrading the phytate. Certain yeasts originating from togwa seem to have developed a natural high phytase production, and P. kudriavzevii TY13 and H. guilliermondii TY14 seem particularly well adapted to phytate degradation in togwa, and is our choice for further studies and strain improvement.

Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models

In this work, we investigated lipid oxidation of cod liver oil during gastrointestinal (GI) digestion using two types of in vitro digestion models. In the first type of model, we used human GI juices, while we used digestive enzymes and bile from porcine origin in the second type of model. Human and porcine models were matched with respect to factors important for lipolysis, using a standardized digestion protocol. The digests were analysed for reactive oxidation products: malondialdehyde (MDA), 4-hydroxy-trans-2-nonenal (HNE), and 4-hydroxy-trans-2-hexenal (HHE) by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS), and for free fatty acids (FFA) obtained during the digestion by gas chromatography-mass spectrometry (GC-MS). The formation of the oxidation products MDA, HHE, and HNE was low during the gastric digestion, however, it increased during the duodenal digestion. The formation of the oxidation products reached higher levels when digestive juices of human origin were used (60 μM of MDA, 9.8 μM of HHE, and 0.36 μM of HNE) [corrected] compared to when using enzymes and bile of porcine origin (0.96, and 1.6 μM of MDA; 0.16, and 0.23 μM of HHE; 0.026, [corrected] and 0.005 μM of HNE, respectively, in porcine models I and II). In all models, FFA release was only detected during the intestinal step, and reached up to 31% of total fatty acids (FA). The findings in this work may be of importance when designing oxidation oriented lipid digestion studies.

Herring and chicken/pork meals lead to differences in plasma levels of TCA intermediates and arginine metabolites in overweight and obese men and women.

Scope: What effect does replacing chicken or pork with herring as the main dietary source of protein have on the human plasma metabolome? Method and results: A randomised crossover trial with 15 healthy obese men and women (age 24–70 years). Subjects were randomly assigned to four weeks of herring diet or a reference diet of chicken and lean pork, five meals per week, followed by a washout and the other intervention arm. Fasting blood serum metabolites were analysed at 0, 2 and 4 weeks for eleven subjects with available samples, using GC‐MS based metabolomics. The herring diet decreased plasma citrate, fumarate, isocitrate, glycolate, oxalate, agmatine and methyhistidine and increased asparagine, ornithine, glutamine and the hexosamine glucosamine. Modelling found that the tricarboxylic acid cycle, glyoxylate, and arginine metabolism were affected by the intervention. The effect on arginine metabolism was supported by an increase in blood nitric oxide in males on the herring diet. Conclusion: The results suggest that eating herring instead of chicken and lean pork leads to important metabolic effects, particularly on energy and amino acid metabolism. Our findings support the hypothesis that there are metabolic effects of herring intake unrelated to the long chain n‐3 polyunsaturated fatty acid content.

Quality of Filleted Atlantic Mackerel (Scomber Scombrus) During Chilled and Frozen Storage: Changes in Lipids, Vitamin D, Proteins, and Small Metabolites, including Biogenic Amines

ABSTRACT Quality changes of vacuum-packed Atlantic mackerel (Scomber scombrus) fillets during 12 months’ frozen storage at −27°C and 9 days’ chilled storage at +4°C were evaluated. Freezing at −27°C preserved the long chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs), both in light and dark muscle, vitamin D, and the low molecular weight metabolites (LMW) (studied by high resolution nuclear magnetic resonance spectroscopy, HR NMR). Protein oxidation took place, especially between 1 and 7 months, decreasing water holding capacity and protein extractability. During chilled storage, no lipid or protein oxidation was observed, but lipolysis increased, and several LMW metabolites relevant for sensory and nutritional quality degraded into non-favorable compounds. The content of biogenic amines was high at day 9 (e.g., 18 mg histamine/100 g), jeopardizing safety. Preservation of mackerel fillets by freezing at −27°C is thus a better option compared to prolonged chilled storage at +4°C; the quality was well preserved for 12 months’ frozen storage.