Phytase in non-ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors.

This review focuses on phytase functionality in the digestive tract of farmed non-ruminant animals and the factors influencing in vivo phytase enzyme activity. In pigs, feed phytase is mainly active in the stomach and upper part of the small intestine, and added phytase activity is not recovered in the ileum. In poultry, feed phytase activities are mainly found in the upper part of the digestive tract, including the crop, proventriculus and gizzard. For fish with a stomach, phytase activities are mainly in the stomach. Many factors can influence the efficiency of feed phytase in the gastrointestinal tract, and they can be divided into three main groups: (i) phytase related; (ii) dietary related and (iii) animal related. Phytase-related factors include type of phytase (e.g. 3- or 6-phytase; bacterial or fungal phytase origin), the pH optimum and the resistance of phytase to endogenous protease. Dietary-related factors are mainly associated with dietary phytate content, feed ingredient composition and feed processing, and total P, Ca and Na content. Animal-related factors include species, gender and age of animals. To eliminate the antinutritional effects of phytate (IP6), it needs to be hydrolyzed as quickly as possible by phytase in the upper part of the digestive tract. A phytase that works over a wide range of pH values and is active in the stomach and upper intestine (along with several other characteristics and in addition to being refractory to endogenous enzymes) would be ideal.

Solid-state fermentation of Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in Jatropha seed cake using Aspergillus versicolor CJS-98.

This study focused on the solid-state fermentation of Jatropha seed cake (JSC), a byproduct generated after biodiesel production. Presence of anti-nutritional compounds and toxins restricts its application in livestock feed. The disposal of the JSC is a major environmental problem in the future, due to the generation of huge quantity of JSC after biodiesel extraction. Hence the JSC was assessed for its suitability as substrate for production and optimization of lipase and protease from Aspergillus versicolor CJS-98 by solid-state fermentation (SSF). The present study was also focused on the biodetoxification of anti-nutrients and toxins in JSC. The SSF parameters were optimized for maximum production of lipase and protease. Under the optimized conditions, the JSC supplemented with maltose and peptone (2%), adjusted to pH 7.0, moisture content 40%, inoculated with 1 × 10(7) spores per 5 g cake and incubated at 25°C, produced maximum lipase, 1288 U/g and protease, 3366 U/g at 96 h. The anti-nutrients like phytic acid (6.08%), tannins (0.37%), trypsin inhibitors (697.5 TIU/g), cyanogenic glucosides (692.5 µg/100 g), and lectins (0.309 mg/ml), were reduced to 1.70%, 0.23%, 12.5 TIU/g, 560.6 µg/100 g and 0.034 mg/ml respectively. The main toxic compound phorbol esters content in the JSC was reduced from 0.083% to 0.015% after SSF. Our results indicate that viability of SSF to utilize the huge amount of seed cake generated after extraction of biodiesel, for production of industrial enzymes and biodetoxification of anti-nutrients, toxins.

Combined phospho- and glycoproteome enrichment in nephrocalcinosis tissues of phytate-fed rats.

RATIONALE: Protein post-translational modifications (PTMs) are directly involved in protein function and cellular activities. Among them, glycosylation and phosphorylation are particularly important modifications on proteins located at extracellular and intracellular domains, respectively. However, the combined detection using phospho- and glycoproteomics is limited mainly due to protocol differences. METHODS: In this study, we developed a novel method for both phospho- and glycoproteome detection from a single sample batch, in which a titanium dioxide cartridge was used to capture the phosphoproteome, and the flow-through solution was processed for capturing N-linked glycopeptides using hydrazide resin. RESULTS: By using 1 mg of protein from kidney tissue lysates from normal and diseased rats, we concurrently identified 437 glycosites/358 phosphosites and 468 glycosites/369 phosphosites in normal and disease kidneys, respectively, by liquid chromatography/tandem mass spectrometric analysis. CONCLUSIONS: Compared with individual PTM analyses, the combined PTM analysis clearly provides more broad implications for PTMs related to the pathological status and discovery of biomarker candidates. Furthermore, the combined protocol thoroughly showed its advantages in enrichment efficiency and biological interpretation compared with current methods.

Detoxification and anti-nutrients reduction of Jatropha curcas seed cake by Bacillus fermentation.

Jatropha curcas seed cake is a by-product generated from oil extraction of J. curcas seed. Although it contains a high amount of protein, it has phorbol esters and anti-nutritional factors such as phytate, trypsin inhibitor, lectin and saponin. It cannot be applied directly in the food or animal feed industries. This investigation was aimed at detoxifying the toxic and anti-nutritional compounds in J. curcas seed cake by fermentation with Bacillus spp. Two GRAS (generally recognized as safe) Bacillus strains used in the study were Bacillus subtilis and Bacillus licheniformis with solid-state and submerged fermentations. Solid-state fermentation was done on 10 g of seed cake with a moisture content of 70% for 7 days, while submerged fermentation was carried out on 10 g of seed cake in 100 ml distilled water for 5 days. The fermentations were incubated at the optimum condition of each strain. After fermentation, bacterial growth, pH, toxic and anti-nutritional compounds were determined. Results showed that B. licheniformis with submerged fermentation were the most effective method to degrade toxic and anti-nutritional compounds in the seed cake. After fermentation, phorbol esters, phytate and trypsin inhibitor were reduced by 62%, 42% and 75%, respectively, while lectin could not be eliminated. The reduction of phorbol esters, phytate and trypsin inhibitor was related to esterase, phytase and protease activities, respectively. J. curcas seed cake could be mainly detoxified by bacterial fermentation and the high-protein fermented seed cake could be potentially applied to animal feed.

Yogurt protects against growth retardation in weanling rats fed diets high in phytic acid.

The purpose of this study was to determine the effects of adding yogurt to animal diets that were high in phytic acid (PA) and adequate in zinc (38 microg Zn/g). The PA:Zn molar ratio was 60:1. Zinc status was determined by documenting growth and measuring the zinc concentration in bone (tibia) and plasma. For 25 days, six groups (n=6) of Sprague-Dawley weanling rats were fed one of six AIN-76 diets. Half of the diets contained PA. Four of the diets contained yogurt with either active or heat-treated (inactive) cultures added at 25% of the diet. The diets were as follows: (a) AIN, (b) AIN with active yogurt, (c) AIN and inactive yogurt, (d) AIN with PA, (e) AIN with PA plus active yogurt and (f) AIN with PA plus inactive yogurt. Body weight, weight gain and zinc concentration in bone and plasma were measured, and food efficiency ratio was calculated. Rats fed diets with PA and yogurt had normal growth compared to the control group. Growth retardation was evident in the group fed the diet with PA and no yogurt. This group had significantly lower body weight compared to all other groups (P<.05). Rats fed diets with PA, with or without yogurt, had significantly lower zinc concentration in bone and plasma (P<.05). Adding yogurt to diets high in PA resulted in normal growth in weanling rats; however, zinc concentration in bone and plasma was still suboptimal.

Studies on protein characteristics and toxic constituents of Simarouba glauca oilseed meal.

In order to exploit the protein rich (47.7 g/100g) simarouba meal in food/feed, studies were conducted on its chemical composition with emphasis on protein characteristics and toxic constituents. Simarouba meal contained high calcium (143 mg/100g) and sodium (79 mg/100g). Saponins with triterpenoid aglycone (3.7 g/100g), alkaloids (1.01 g/100g), phenolics (0.95 g/100g) and phytic acid (0.73 g/100g) were the major toxic constituents identified in simarouba meal. TLC and HPLC results indicated that among different fractions of simarouba saponins, one dominant fraction accounted for about 28%. Proteins of simarouba recorded high in vitro digestibility (88%). SDS-PAGE revealed four major protein bands in molecular weight ranges of 20-24, 36-45 and 55-66 kDa. Apart from, glutamic acid (23.43 g/100g protein) and arginine (10.75 g/100g protein), simarouba protein contained high essential amino acids like leucine (7.76 g/100g protein), lysine (5.62 g/100g protein) and valine (6.12 g/100g protein). Among nutritional indices, simarouba meal recorded a good EAA Index (75.02), C-PER (1.90) and PDCAAS (1.0-Adult group).

Effect of dietary phytate on magnesium bioavailability and liver oxidant status in growing rats.

Male albino rats (initial average weight 60 g) were fed semi-synthetic diets based on casein, corn starch and sunflower oil over 21 days. All diets were supplemented with 300 mg magnesium from MgSO4x7H2O and 0,7.5 and 15 g phytic acid (PA) from sodium phytate per kg. The addition of PA to the diets resulted in a dose-dependent decrease of apparent Mg absorption and Mg concentration in the plasma and femur. Impaired Mg bioavailability due to 15 g PA/kg diet was accompanied by an increase of hepatic thiobarbituric acid reactive substances and protein carbonyls as well as by a moderate decline in liver reduced glutathione (GSH) levels. The liver homogenates of rats receiving the diets with 7.5 and 15 g PA/kg, respectively, were much more susceptible to iron-induced lipid peroxidation than those of the controls. Hepatic antioxidative enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glucose-6-phosphate dehydrogenase (G6PDH)], alpha-tocopherol concentration and phenyl-N-tert-butylnitrone (PBN) adducts using electron spin resonance spectroscopy remained unchanged by the different dietary treatments. Under the conditions of a marginal dietary Mg supply, phytate had pro-oxidative rather than antioxidative effects in the case of liver metabolism.

Effects of inositol hexasulphate, a casein kinase inhibitor, on dentine phosphorylated proteins in organ culture of mouse tooth germs.

To study the effects of impaired protein phosphorylation on dentine formation and mineralization, inositol hexasulphate, an intracellular type I and type II casein kinase inhibitor, was used in an in vitro organotypic culture system. Mandibular first molar tooth germs were dissected from 18-day-old mouse embryos and cultured for 11 days with and without inositol hexasulphate at different concentrations. At 0.04-0.08 mM inhibitor, cellular alterations were not detected. Dentine displayed the characteristic purple-blue colour when Stains all, a specific stain for extracellular phosphoproteins, was used. At 0.1 mM, dentine failed to stain and mineralization did not occur, as seen from the von Kossa method. The presence of numerous lysosome-like vesicles inside cells indicated that the experiment was at the limits of cytotoxicity; higher concentrations induced severe cellular alterations. Therefore, quantitative radioautography was carried out on germs treated or not with the inhibitor at 0.1 mM. [33P]-phosphate incorporation showed that grain density in inhibited germs compared with that in control germs was about double in odontoblasts and half in the predentine/dentine compartment. In the presence of inositol hexasulphate the incorporation of [3H]serine into odontoblast cell bodies was unchanged between 2 and 24 h while in predentine/dentine, grain density was higher between 1 and 4 h, and reduced at 24 h. Both with [33P]phosphate and [3H]serine, labelling was seen throughout the porous dentine formed in vitro and not as a band located at the predentine/dentine junction, as is the case in vivo. With [3H]proline, in the presence of the inhibitor, a small reduction of grain density occurred in cell bodies, no significant difference was seen between 1 and 4 h in predentine/dentine, and more silver grains were present after 24 h both in cells and in the matrix. The radioautographic data support the view that the inhibitor interacts mostly with post-transductional phosphorylation and does not alter significantly other cell synthetic pathways and functions. Finally, the experiments presented here confirm that phophorylated proteins have a key role in dentine mineralization.

Novel anticancer function of inositol hexaphosphate: inhibition of human rhabdomyosarcoma in vitro and in vivo.

Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that has been shown to suppress the growth of epithelial cancers, including those of breast and colon. The objective of this study was to investigate whether IP6 inhibits growth of rhabdomyosarcoma (RMS), a tumor of mesenchymal origin, which is the most common soft tissue sarcoma in children. We performed both in vitro and in vivo studies to evaluate the effect of IP6 on human RD cells growth. Our results show that IP6 suppresses growth of rhabdomyosarcoma cell line (RD) in vitro in a dose-dependent fashion. A 50% inhibition of cell growth (IC50) was induced by < 1.0 mM IP6. However, the removal of IP6 from the media, after 72 hours of treatment, allowed cells to recover their logarithmic growth. Exposure of RD cells to IP6 led to differentiation; cells became larger with abundant cytoplasm, expressing higher levels of muscle-specific actin. Consistent with in vitro observation, IP6 suppressed RD cell growth in vivo, in a xenografted nude mice model. When compared to controls, IP6-treated mice produced a 25 fold smaller tumors (p = 0.008), as observed after a two weeks treatment. In a second experiment, wherein the treatment period was extended to five weeks, a 49 fold (p = 0.001) reduction in tumor size was observed in mice treated with IP6. Histologically no evidence of tumor cell necrosis was observed. These data suggest a potential usefulness of this cytostatic, and non-cytotoxic, compound in novel therapeutic strategies for these types of tumor.

IP6 in treatment of liver cancer. I. IP6 inhibits growth and reverses transformed phenotype in HepG2 human liver cancer cell line.

Hepatocellular carcinoma (HCC) is a common tumor world-wide with extremely poor prognosis. Recent studies have shown that inositol hexaphosphate (IP6), a naturally occurring carbohydrate, has novel anti-cancer function in various in vitro and in vivo models. The aim of this study was to assess whether IP6 could inhibit the growth of human hepatocellular carcinoma. We treated HepG2, a human liver cancer cell line in vitro with IP6 and evaluated its effect on growth and differentiation. IP6 treatment of HepG2 cells caused a dose-dependent growth inhibition. Compared to other cancer cell lines, HepG2 cells were quite sensitive to IP6, IC50 (50% inhibition of cell growth) of IP6 being < 1.0 mM (0.338 mM). Treatment with IP6 decreased the ability of HepG2 cells to form colonies, as assessed in the plating efficiency assay. Morphological changes induced by IP6 were consistent with differentiation of HepG2 cells. Exposure of HepG2 cells to IP6 drastically decreased the rate of production of alpha-fetoprotein (AFP), a tumor marker of HCC, indicating also that IP6 treatment leads to differentiation of malignant liver cells. Further, IP6 treatment caused a decreased expression of mutant p53 protein in HepG2 cells, with no significant change in the expression of wild-type p53. The expression of p21WAF1 protein was increased by 1.5 fold, as determined by immunocytochemical staining and ELISA assay. These data demonstrate that IP6 inhibits the growth, and induces differentiation, and a less aggressive phenotype of HepG2 cells, suggesting a role of IP6 in the treatment of HCC.

Neuronal cytotoxicity of inositol hexakisphosphate (phytate) in the rat hippocampus.

D-myo-Inositol hexakisphosphate (InsP6, phytate), a normal cellular constituent, was found to be toxic to neuronal perikarya when injected into the rat hippocampus. However, the extrinsic cholinergic innervation of the hippocampus (as estimated by staining for acetylcholinesterase) was unaffected. Its potency as a toxin was approximately equal to that of the excitotoxin quinolinate. Other highly charged derivatives of inositol (inositol hexakissulphate, inositol monophosphate) were not toxic. The cytotoxicity of InsP6 was not due to a high osmolality, or to seizure-induced lesions, but was reduced by calcium. Nevertheless, the toxicity was not due to chelation of brain calcium by InsP6, as another calcium chelator with a higher affinity for calcium, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), produced only a very mild lesion. Thus, abnormal metabolism of InsP6 might possibly contribute to neuronal death in neurodegenerative diseases.

Modification of N-butyl-N-(4-hydroxybutyl)nitrosamine-initiated urinary bladder carcinogenesis in rats by phytic acid and its salts.

The effects of dietary phytic acid and its salts on the promotion stage of two-stage urinary bladder carcinogenesis were examined. Male F344 rats were initiated by exposure to 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water for 4 wk, and then treated with basal diet containing a 2% supplement of phytic acid (PA), phytic acid dodecasodium salt (Na-PA), phytic acid dodecapotassium salt (K-PA), phytic acid hexamagnesium salt n-hydrate (Mg-PA) or no added chemical for 32 wk. Na-PA significantly increased the development of preneoplastic and neoplastic lesions of the urinary bladder. K-PA also brought about a tendency for increase in papillomas, whereas Mg-PA and PA were without effect. Both Na-PA and K-PA caused elevation of urinary pH, and Na+ or K+ concentration, respectively. These results confirm the promoting activity of the sodium salt of phytic acid for urinary bladder carcinogenesis and indicate modulation by urinary components, as demonstrated by increases in urinary pH, and Na+ concentration.

[The effect of a supplement of microbial phytase on zinc availability]. / Effekt einer Zulage mikrobieller Phytase auf die Zinkverfügbarkeit.

A study of 35 (5 x 7) male, individually housed, albino rats (initial average weight = 50 g) was undertaken to examine the effect of an addition of microbial phytase to a diet containing phytate on the availability of zinc. The rats were fed a semisynthetic diet based of egg white and cornstarch over a 3-week period. All diets were supplemented with 20 mg Zn/kg. Group I (control) was fed the basal diet free of phytic acid (PA) and phytase. By replacing cornstarch by Na-phytate (0.5% in group II and 1.0% group III), molar phytate: Zn ratios of 25 and 50:1 were obtained, respectively. In groups IV (0.5% PA) and V (1.0% PA) 1000 U of microbial phytase were added. A molar phytate:Zn ratio of 25 (group II) and 50:1 (group III) resulted in a dose-dependent depression of growth and feed efficiency ratio. These negative effects of the addition of PA could be completely counteracted by the supplementation of 1,000 U of phytase in group IV and partially so in group V. Similarly, the apparent absorption and retention of Zn, Zn-concentration in femur and testes and different Zn-status-parameters in plasma (Zn-concentration, percent unsaturated plasma-Zn binding capacity, activity of alkaline phosphatase) were improved by adding 1,000 U microbial phytase/kg diet. The present study shows that an addition of microbial phytase to phytate-rich diets considerably improves the availability of Zn in growing rats.

Carcinogenicity study in rats of phytic acid 'Daiichi', a natural food additive.

The carcinogenicity of phytic acid 'Daiichi' (PA), a natural food additive, was examined in Fischer 344 rats of both sexes. PA was added to the drinking-water of groups of 60 male and 60 female rats at levels of 1.25 or 2.5% for 100-108 wk. There was a dose-dependent reduction in the mean final body weights of rats treated with PA. Necrosis and calcification of the renal papillae were observed in PA-treated rats, but not in the controls. The incidences of necrosis (calcification) were as follows: one (three) out of 57 males given 2.5% PA; one (none) out of 59 males given 1.25% PA; 10 (17) out of 55 females given 2.5% PA; six (six) out of 58 females given 1.25% PA. Renal papillomas occurred in three of the high-dose male rats, four of the high-dose female rats, and three of the low-dose female rats. The development of papillomas seemed to be related to calcification and necrosis of the renal papillae induced by PA. While many other tumours developed in all groups, including the controls, the organ distribution of these neoplasms and their histological characteristics did not differ significantly from those known to occur spontaneously in this strain of rats.

The influence of infusion rate on the acute intravenous toxicity of phytic acid, a calcium-binding agent.

The intravenous toxicity of phytic acid (inositol hexakisphosphate, IHP) has recently become of interest because of the potential for IHP incorporation into red blood cells to achieve a therapeutically useful shift in the hemoglobin-oxygen dissociation curve (Gersonde, K. and Nicolau, C., Blut, 39 (1979) 1). The observed acute intravenous toxicity of IHP in rodents is consistent with its recognized capacity to bind calcium. The toxic manifestations of intravenous IHP are a function of rate of infusion as well as total dose, with some seemingly anomalous variations which may related to compensatory mechanisms. The data suggest that significant alterations of plasma calcium and the toxic potential of such alterations are not likely to result from the administration of red blood cells with IHP incorporated.