Monitoring the stepwise phytate degradation in the upper gastrointestinal tract of pigs

The degradation and formation of inositol phosphates as affected by microbial phytase and gastrointestinal enzyme activities during the passage of phytate through the stomach and small intestine were studied in two experiments with four barrows and three collection periods. The degradation and formation of inositol phosphates were measured at the duodenal and ileal sites using Cr‐NDR, TiO₂ and Co‐EDTA as indigestible markers. In experiment 1, the effect of graded doses of Aspergillus niger phytase (0, 150 and 900 FTU Natuphos^® kg^?1), added to a maize–soybean meal‐based diet with very low intrinsic phytase activity on the degradation of phytate and the formation of inositol phosphates during digestion in the stomach and small intestine was investigated. In experiment 2, three different mixtures of inositol phosphates, produced by Aspergillus niger phytase, containing mainly high, intermediate and low phosphorylated inositol phosphates, were added to the same maize–soybean meal‐based diet as used in experiment I. The fate of the inositol phosphates during digestion in the stomach and small intestine was studied. Experiment 1 showed that the extent of phytate degradation was dependent of the graded dietary phytase activities. At high phytase activity (900 FTU kg^?1 of diet), strong phytate degradation occurred and the once hydrolysed phytate was rapidly dephosphorylated to lower inositol phosphates (mainly inositol di‐ and triphosphates). Intermediate inositol phosphates, such as inositol tetraphosphates, were quantitatively unimportant in duodenal and ileal digesta. At a phytase activity of 150 FTU kg^?1 of diet, a broader spectrum of intermediate inositol phosphates was determined, which was probably due to a slower breakdown of phytate. Experiment 2 showed as a predominant result that lower inositol phosphates such InsP₄ and InsP₃ were degraded, whereas InsP₂ accumulated in the duodenal and ileal digesta. No substantial disappearance of phytate from the stomach and small intestine was found when high concentrations of soluble phytate were added to the diet, which indicates that no substantial phytate absorption occurs in the upper part of the pig gut. Copyright © 2005 Society of Chemical Industry     

Application of phytases from bifidobacteria in the development of cereal-based products with amaranth

The effects of the inclusion of purified phytases from Bifidobacterium longum spp. infantis ATCC15697 and Bifidobacterium pseudocatenulatum ATCC27919 on phytate (InsP ₆) levels were analyzed during breadmaking process. Two different levels of whole amaranth (Amaranthus cruentus) flour (25 and 50 %) were used in bread dough preparation, and they were compared to control doughs made with 100 % wheat flour and 100 % whole wheat flour. Bread samples made with 50 % of amaranth flour showed a significant decrease in technological quality parameters in comparison with control white breads. However, a 25 % of amaranth flour improved the nutritional value of the bread, with only a slight depreciation in the quality. Addition of bifidobacterial phytases resulted in higher InsP ₆ degradation compared with a commercial fungal phytase, without affecting the bread quality. InsP ₆ reduction was especially efficient in breads with 25 % amaranth, leading to InsP ₆ levels below the threshold of mineral bioavailability inhibition for Fe and Zn in human nutrition.     

Purification and characterization of a phytate-degrading enzyme from germinated oat (Avena sativa)

A phytate-degrading enzyme (myo-inositol hexakisphosphate phosphohydrolase) has been purified about 5,400-fold from germinated oat seedlings to apparent homogeneity. The molecular mass of the native monomeric enzyme was estimated to be about 67 kDa. Optimal pH for degradation of phytate was 5.0 and the optimal temperature 38 °C. Kinetic parameters for the hydrolysis of Na-phytate are K_M 30 µM and k_cat 356 s⁻¹ at 35 °C and pH 5.0. The oat phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyses phytate in a stepwise manner. The first hydrolysis product was identified as D /L -l(1,2,3,4,5) P₅. © 1999 Society of Chemical Industry     

Myo-inositol phosphates profile of buckwheat and quinoa seeds: Effects of hydrothermal processing and solid-state fermentation with Rhizopus oligosporus

Phytate content, bioavailability and myo-inositol phosphates profile were analysed in pseudocereals (i.e., white and coloured quinoa, buckwheat) subjected to thermal processing and solid-state fermentation with Rhizopus oligosporus. Fair buckwheat groats were characterised by the highest level of phytate (18.3 µmol/g), with 54% share of InsP₆ fraction. Processing (cooking, roasting, tempe-type fermentation) resulted in the significant decrease in phytate content, its bioavailability and the share of higher phosphorylated forms of myo-inositol (InsP_6-5) in the profile of all the samples. Fermentation of cereals resulted in generation of high amounts of inositol triphosphates, particularly in the case of buckwheat (32% share in the whole profile). The path of dephosphorylation, identified after fermentation of quinoa seeds including intermediate products – Ins(1,2,4,5,6)P₅ and Ins(1,2,6)P₃ – confirmed that the dominant phytase activity could be attributed to the presence of microbial 3-phytases from R. oligosporous.     

STEREOSPECIFICITY OF MY0-INOSITOL HEXAKISPHOSPHATE DEPHOSPHORYLATION BY PHYTATE-DEGRADING ENZYMES OF CEREALS

Using a combination of High‐Performance Ion Chromatography analysis and kinetic studies the stereospedfidty of myo‐inositol hexakisphosphate dephosphorylation by the spelt phytase D21, the rye phytase, the barley phytases P1 and P2, and the oat phytase was established. The data demonstrate that all the cereal phytate‐degrading enzymes under investigation dephosphorylate myo‐inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D‐Ins(l,2, 3,5, 6)P₅, D‐Ins(l,2, 5,6)P₄, D‐Ins(l,2, 6)P₃, D‐Ins(l,2)P₂ to finally Ins(2)P (notation 4/3/5/6/1).     

Effect of natural fermentation on the content of inositol phosphates in lentils

The effects of natural fermentation upon phytic acid and less phosphorylated inositol phosphates ofLens culinaris var vulgaris cultivar Magda-20 were investigated. Seven fermentation runs were made following a 2² complete factorial design with three replicated centre points to study the effect of different conditions of temperature (28, 35 and 42°C) and broth concentration (79, 150 and 221 g/l). Samples were taken for each of them at daily intervals (0, 24, 48, 72 and 96 h). The pH value declined sharply in the first 24 h of fermentation, becoming stabilized from this time. The relation between lactic acid and titratable acidity presented important differences between the different fermentations, ranging from 30–80%. Phytic acid (IP ₆), inositol pentakis (IP ₅), tetrakis (IP ₄) and tris-(IP ₃) phosphates were quantitatively determined. The content of total inositol phosphates showed a maximum reduction of 63% at 72 h under the fermentation conditions of 42°C and 79 g/l.     

Selection of lactic acid bacteria with high phytate degrading activity for application in whole wheat breadmaking

Whole wheat products although highly recommended from the nutritional point of view, contain high levels of phytic acid, an antinutrient that decrease the mineral bioavailability. The objective of this study was to select strains with high phytate-degrading activity from different parts of the gastrointestinal tract of chickens following a phytate-rich diet and to test their suitability for the breadmaking process of whole wheat bread. Different lactobacilli and bifidobacteria strains were isolated and individually assayed for phosphatase and phytase activities, since both enzymes could contribute to the degradation of phytate. The isolates showing the highest phytate degrading activity belonged to the species Bifidobacterium dentium, Lactobacillus reuteri (L-M15) and Lactobacillus salivarius (L-ID15). The two lactobacilli L-M15 and L-ID15 were selected and tested their fermentative ability in whole wheat breadmaking. Whole wheat breads in the presence of the selected lactobacilli had similar technological quality than the control (in absence of lactobacilli) and extended freshness; moreover, their presence resulted in bread crumbs with lower levels of inositol phosphates. Overall, the two intestinal lactobacilli strains showing high phytate degrading activity were proven to have good properties for being used as starters in whole wheat breadmaking process.     

Phytate degradation by Bifidobacterium on whole wheat fermentation

Whole cereal-based products have a beneficial effect on health, but they also contain high levels of phytate, defined as anti-nutrient. The possible use of different strains of bifidobacterial species (B. catenulatum, B. longum and B. breve) with phytate degrading activity as starters in the fermentation process was investigated. Fermentative parameters of doughs (pH, volume, total titrable acidity [TTA] and acetic and lactic acids production) and the hydrolysis profile of the phytic acid were determined during whole wheat dough fermentation. During fermentation in the presence of different bifidobacterial strains, the concentration of phytic acid showed a progressive decrease, leading to a higher release of hydrolysis products than in the control, within short fermentation time. According to the fermentative parameters bifidobacterial strains showed a good adaptation to the dough ecosystem. The range of TTA and lactic acid production was dependent on the strain. Among the tested bifidobacterial strains, those belonging to the species B. breve and B. longum induced a higher hydrolysis of phytic acid with simultaneous production of lower inositol phosphates. In addition, dough containing those strains had high pH and minor acidity than those containing a commercial starter (L. plantarum). Therefore, the tested bifidobacterial strains could be used as breadmaking starters contributing to different acidification degrees and promoting simultaneously the degradation of phytic acid in the whole wheat dough.     

The ability of <Emphasis Type="Italic">Lactobacillus</Emphasis> adhesin EF-Tu to interfere with pathogen adhesion

Probiotic bacteria play an important role in preventing widespread colonization of enteropathogens in human gastrointestinal tract. Lactobacillus plantarum CS24.2, isolated from child fecal sample, and L. rhamnosus GG, a widely accepted commercial probiotic strain, were examined in vitro for their ability to inhibit the colonization of enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhi (S. Typhi) on human intestinal epithelial cell line (Caco-2). Different adhesion assays such as competitive inhibition, adhesion inhibition, and displacement were carried out for the assessment of antagonistic activity of probiotic bacteria toward adhesion of enteropathogens to Caco-2 cells. Both the probiotic bacteria were able to inhibit pathogen colonization between 30–90% under different assay conditions. The gene coding for the known Lactobacillus adhesion factor—elongation factor Tu (EF-Tu)—was amplified from L. plantarum CS24.2 and cloned into E. coli expression vector, pET30(a). The partially purified recombinant EF-Tu was able to inhibit 50% adhesion of L. plantarum CS24.2 to Caco-2 cells but there was no effect on L. rhamnosus GG adhesion under competitive inhibition assay. To investigate the functional role of lactobacilli EF-Tu in pathogen inhibition to Caco-2 cells, competitive adhesion assay was carried out and there was significant reduction in the adhesion of E. coli (35.3%) and S. Typhi (47.7%) to Caco-2 cells.     

蜂粮中Lactobacillus kunkeei的益生特性研究

蜂粮是植物花粉、蜂蜜和蜜蜂唾液的发酵混合物,是营养丰富的天然食材。作者从蜂粮中分离到61株乳酸杆菌,经16S rRNA基因测序,鉴定为Lactobacillus kunkeei。首先,用邻苯二甲醛法对61株L.kunkeei进行胆固醇去除能力检测,其中5株菌胆固醇去除能力均大于15%,菌株B35对胆固醇的去除率达到(29.07±1.30)%。随后,对这5株L.kunkeei进行酸耐受性、胆盐耐受性、抗生素耐药性及抑菌等益生特性进行研究。耐酸实验和耐胆盐实验结果表明,5株L.kunkeei均具有良好的耐酸性和胆盐耐受性。药敏试验结果表明,5株L.kunkeei对4种临床常用抗生素(红霉素、克林霉素、庆大霉素、万古霉素)均敏感。通过琼脂扩散法抑菌实验,发现5株L.kunkeei对大肠埃希氏菌、金黄色葡萄球菌均有一定的抑菌作用。B35菌株的细胞黏附性能良好,对Caco-2细胞的黏附率达到3.32%。从蜂粮中分离到5株具有良好益生特性的菌株,其中菌株B35的益生特性最佳,为益生菌的开发与利用提供了新的菌种资源。     

Effects of Inositol Tri-, Tetra-, Penta-, and Hexaphosphates on In Vitro Estimation of Iron Availability

The effects of inositol tri-, tetra-, penta-, and hexaphosphates on iron solubility were studied in vitro. Pure inositol phosphates (0.125–10 μmol) were added to 1g of freeze-dried wheat roll and digestion was performed simulating physiological conditions. Soluble iron was used as an index of iron bioavailability. The pure inositol phosphate fractions were prepared by hydrolysis of sodium phytate and separation on anion exchange resin. The inositol phosphates were identified and quantified by HPLC. Addition of 10 μmol inositol hexa- and pentaphpsphate reduced iron solubility from 39% (reference) to 0.2% and 6%, respectively, while the same amount of inositol tetra- and tri-phosphate slightly increased the iron solubility. Thus, degradation of inositol hexa- and pentaphosphates seemed to significantly reduce the inhibiting effect on iron availability.     

Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products

The yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus are associated with fermentation of West African indigenous foods. The aim of this study was to characterize potential probiotic properties of S. cerevisiae and K. marxianus isolates from the West African milk products lait caillé and nunu and a cereal-based product mawè. The strains (14 in total) were identified by 26S rRNA gene sequencing and characterized for survival at gastrointestinal stress (bile salts and low pH) and adhesion to Caco-2 intestinal epithelial cells. Selected yeast isolates were tested for their effect on the transepithelial electrical resistance (TEER), using the intestinal epithelial cell line Caco-2 and for maintenance of intracellular pH (pH_i) during perfusion with gastrointestinal pH (3.5 and 6.5). All tested yeasts were able to grow in bile salts in a strain-dependent manner, exhibiting a maximum specific growth rate (μ_max) of 0.58–1.50 h⁻¹. At pH 2.5, slow growth was observed for the isolates from mawè (μ_max of 0.06–0.80 h⁻¹), whereas growth of yeasts from other sources was mostly inhibited. Yeast adhesion to Caco-2 cells was strain specific and varied between 8.0% and 36.2%. Selected strains of S. cerevisiae and K. marxianus were able to maintain the pH_i homeostasis at gastrointestinal pH and to increase TEER across the Caco-2 monolayers, indicating their potential to improve intestinal barrier functions. Based on overall results, strains of K. marxianus and S. cerevisiae from mawè exhibited the highest probiotic potential and might be recommended for further development as starter cultures in West African fermented products.     

Probiotic potential of <Emphasis Type="Italic">L. sake</Emphasis> C2 isolated from traditional Chinese fermented cabbage

The probiotic properties of Lactobacillus sake C2 isolated from traditional Chinese fermentation cabbage were evaluated. L. sake C2 displayed good tolerance to the gastrointestinal environments including low pH, presence of bile salts, pepsin, and pancreatin. L. sake C2 not only could produce the bacteriocin with a broad inhibitory spectrum, but also secrete a large amount of lactic acid (11.26 mg/mL) and certain amount of H₂O₂ (3.14 mg/mL). L. sake C2 displayed a high adhesion rate of 15.2 ± 1.2% to Caco-2 cells. Moreover, viable cells of L. sake C2 have fairly strong ability to remove cholesterol (53.2 ± 1.4%). These results indicated that L. sake C2 has a potential as probiotic. Moreover, this study suggested that besides lactic acid bacteria originating from human, those isolated from fermented foods have potential to be used as probiotic and may be exploited in food industry and probiotic preparations.     

Natural Fermentation of Lentils: Influence of Time,Concentration and Temperature on the Kinetics of Hydrolysis of Inositol Phosphates

Lentil (Lens culinaris var vulgaris) flour was naturally fermented for 96 h at various conditions of concentration (79, 150 and 221 g litre⁻¹) and temperature (28°C, 35°C and 42°C). The content of total inositol phosphates (IP-total) and individual inositol phosphates (hexa- (IP₆), penta- (IP₅), tetra- (IP₄) and tri- (IP₃) phosphates) were analysed to establish the changes of these compounds during natural fermentation of lentils. The preparation of the lentil suspension brought about 16–27% reduction of the total inositol phosphates. At the end of 96 h of natural fermentation maximum IP loss (70–75%) was achieved for an experiment carried out at minimum concentration. For IP₆, the largest decrease was achieved at the highest temperature, the fermentation condition that also brought about the highest IP₅ content.     

Effect of Several Germination Treatments on Phosphatases Activities and Degradation of Phytate in Faba Bean (Vicia faba L.) and Azuki Bean (Vigna angularis L.)

Abstract: Two assays were conducted to investigate the changes of faba bean (Vicia faba L.) and azuki bean (Vigna angularis L.) phosphatases (phytase [Phy] and acid phosphatase [AcPh]) and the degradation of its substrates (inositol phosphate esters) during seed germination. The 1st assay was to establish the optimal germination conditions of faba bean and azuki bean to improve the endogenous phosphatases and increase the hydrolysis of phytate and, in the second assay, to determine the different lower phosphate esters of myo‐inositol produced during the germination process. In the 1st assay, seeds were soaked for 12 and 24 h and germinated for 3 and 5 d with and without the addition of gibberellic acid (GA₃). In the second assay, seeds were soaked for 12 h and germinated for 1, 3, and 5 d with GA₃. Phy (up to 3625 and 1340 U/kg) and AcPh (up to 9456 and 2740 U/g) activities, and inositol hexaphosphate (IP6) (8.23 and 7.46 mg/g), inositol pentaphosphate (IP5) (0.55 and 0.82 mg/g), and inositol tetraphosphate (IP4) (0.26 and 0.01 mg/g) were detected in ungerminated faba bean and azuki bean, respectively. The germination process caused a significant increase of Phy and AcPh activities in faba bean (up to 147% and 210%) and azuki bean (up to 211% and 596%) and a reduction in the phytate phosphorus content (up to 81% and 63%, respectively). Phytate phosphorus content was affected only by soaking time in the case of faba bean. Finally, during the course of germination, IP6 and IP5 were rapidly degraded in faba bean (88% and 39%) and azuki bean (55% and 56%), and IP4 was only a short‐living intermediate, which was increased during hydrolysis and degraded to inositol triphosphate. In this manner we could obtain a low‐phytate, endogenous phosphatase‐rich ingredient for enhancing human nutrition.     

Changes in quantities of inositol phosphates during maturation and germination of legume seeds

 The changes in the quantities of inositol phosphates during the maturation and germination of pea, faba bean and lupin seeds were determined in two consecutive (1993 and 1994) years of differing weather conditions. Irrespective of the year, all seeds accumulated predominantly inositol hexaphosphate (IP₆). The weather conditions influenced the accumulation of inositol phosphates in maturing seeds, but they did not influence the total content. Gradual degradation of inositol phosphates occurred during seed germination. After 8 days of germination, IP₆ was degraded by some 80% in peas, 78% in faba beans and 42% in lupin seeds. The enzymic hydrolysis of higher forms of inositol phosphates (IP₆ and IP₅) in germinating seeds was assumed to yield inositol tetraphosphate (IP₄) and inositol triphosphate (IP₃), because the quantities of these compounds increased during seed germination. Received: 1 August 1997 / Revised version: 13 October 1997     

Changes in quantities of inositol phosphates during maturation and germination of legume seeds

 The changes in the quantities of inositol phosphates during the maturation and germination of pea, faba bean and lupin seeds were determined in two consecutive (1993 and 1994) years of differing weather conditions. Irrespective of the year, all seeds accumulated predominantly inositol hexaphosphate (IP₆). The weather conditions influenced the accumulation of inositol phosphates in maturing seeds, but they did not influence the total content. Gradual degradation of inositol phosphates occurred during seed germination. After 8 days of germination, IP₆ was degraded by some 80% in peas, 78% in faba beans and 42% in lupin seeds. The enzymic hydrolysis of higher forms of inositol phosphates (IP₆ and IP₅) in germinating seeds was assumed to yield inositol tetraphosphate (IP₄) and inositol triphosphate (IP₃), because the quantities of these compounds increased during seed germination. Received: 1 August 1997 / Revised version: 13 October 1997     

Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use

A set of simple in vitro tests (identification by species-specific PCR, genetic diversity, phage sensitivity, growth and viability in milk, resistance to salts and flavor compounds, bacterial interactions, tolerance to simulated gastric juice and bile, bile salts deconjugation, hydrophobicity and β-galactosidase and antibacterial activities), that can be carried out in almost every laboratory of microbiology, mainly in developing countries where there is often limited access to sophisticated techniques, allowed us to identify, among 19 intestinal human isolates, a potential candidate for new probiotic dairy foods for the local market. Lactobacillus gasseri LgF_37/1 performed well in the culture media used for the enumeration of probiotic bacteria in argentinian dairy products. This strain showed also high tolerance to the technological challenges assessed, bile salts resistance, the capacity to produce bacteriocin-like metabolites, to inhibit pathogenic bacteria, to deconjugate bile salts and high hydrophobicity. Further in vivo research should be carried out with this strain before claiming probiotic properties for it. However, the use of a set of simple in vitro techniques proved to be important to determine which strains should undergo future and more complex studies.     

Influence of fermentation on the nutritional value of two varieties of<Emphasis Type="Italic"> Vigna sinensis</Emphasis>

Cowpeas ( Vigna sinensis) are a good source of protein and energy. They are widely consumed all over the world, mainly in rural populations, and satisfy a considerable proportion of the protein requirements. However, like other pulses, cowpeas contain several antinutritional factors, which limit their consumption and affect the digestibility and bioavailability of nutrients. Fermentation seems to enhance the nutritive value of legumes. The present work studies the effect of natural fermentation and subsequent cooking process of two varieties of Vigna sinensis on the content of protein, minerals, ash, fat, soluble and insoluble dietary fibre, -galactosides, inositol phosphates, trypsin inhibitor activity (TIA), water-soluble vitamins (thiamin and riboflavin) and protein quality parameters, in vivo digestibility and PER (protein efficiency ratio). The processes applied caused a significant increase of vitamin B_2, and significant reductions in the contents of TIA (40%) and phytates (100%), main antinutritional factors present in cowpeas. Likewise, the soluble fibre and -galactosides compounds that produce flatulence diminished (67 and 100%, respectively). Protein digestibility for Orituco variety was adequate among legumes (82.8–88.4%), although only Vigna sinensis var. Orituco showed an acceptable PER value (1.63).     

Water extractable phytochemicals from Capsicum pubescens (tree pepper) inhibit lipid peroxidation induced by different pro-oxidant agents in brain: in vitro

This study seeks to determine the antioxidant and neuroprotective properties of aqueous extract of ripe and unripe Capsicum pubescens (tree pepper) on some pro-oxidant induced lipid peroxidation in rat’s brain (in vitro). The total phenol, vitamin C, ferric reducing antioxidant property (FRAP) and Fe (II) chelating ability of the extracts of C. pubescens were determined. Thereafter, the ability of the extracts to inhibit lipid peroxidation (induced by FeSO₄, sodium nitroprusside or quinolinic acid) in rat’s brain homogenates (in vitro) was determined. The results of the study revealed that ripe C. pubescens had a significantly higher (P < 0.05) total phenol content [ripe (113.7 mg/100 g), unripe (70.5 mg/100 g)] and reducing power than the unripe pepper. However, there was no significant difference (P > 0.05) in the vitamin C [ripe (231.5 μg/g), unripe (224.4 μg/g)] content and Fe (II) chelating ability of the extracts. However, both extracts significantly (P < 0.05) inhibited lipid peroxidation induced by the pro-oxidant agents [25 μM Fe(II), 7 μM sodium nitroprusside and 1 mM quinolinic acid] in the rat’s brain homogenates in a dose-dependent manner. Nevertheless, the ripe pepper extracts inhibited MDA (Malondialdehyhide) production in the rat’s brain homogenates than the unripe pepper. Conversely, both extracts did not significantly (P > 0.05) inhibit Fe (II)/H₂O₂ induced decomposition of deoxyribose. It was therefore concluded that ripe and unripe C. pubescens would inhibit lipid peroxidation in rat brain in vitro. However, the ripe pepper was a more potent inhibitor of lipid peroxidation in the rat’s brain; this is probably due to its higher phenol content and reducing power.