Fermentation of food and feed: A technology for efficient utilization of macro and trace elements in monogastrics

Mineral deficiencies, especially of iron, zinc, and calcium, respectively, negatively affect human health and may lead to conditions such as iron deficiency anemia, rickets, osteoporosis, and diseases of the immune system. Cereal grains and legumes are of global importance in nutrition of monogastrics (humans and the respective domestic animals) and provide high amounts of several minerals, e.g., iron, zinc, and calcium. Nevertheless, their bioavailability is low. Plants contain phytates, the salts of phytic acid, chemically known as inositol-hexakisphosphate, which interact with several minerals and proteins. However, phytate may be hydrolysed by phytase. This enzyme is naturally present in plants and also widely distributed in microorganisms. Several food processing methods have been reported to enhance phytate hydrolysis, due to the activation of endogenous phytase activity or via the enzyme produced by microbes. In recent years, fermentation for food and feed improvement and preservation, respectively, has gained increasing interest as a promising method to degrade phytate and enhance mineral utilization in monogastrics. Indeed, several in vitro as well as in vivo studies confirm a positive effect on the utilization of minerals, such as P, Ca, Fe and Zn, using sourdough fermentation for baking or fermentation of legumes, mainly soybeans. This review summarizes the current knowledge regarding the potential of fermentation to enhance macro and trace element bioavailability in monogastric species.     

Bioavailability of iron, zinc, phytate and phytase activity during soaking and germination of white sorghum varieties

The changes in phytate, phytase activity and in vitro bioavailability of iron and zinc during soaking and germination of three white sorghum varieties (Sorghum bicolor L. Moench), named Dorado, Shandweel-6, and Giza-15 were investigated. Sorghum varieties were soaked for 20 h and germinated for 72 h after soaking for 20 h to reduce phytate content and increase iron and zinc in vitro bioavailability. The results revealed that iron and zinc content was significantly reduced from 28.16 to 32.16% and 13.78 to 26.69% for soaking treatment and 38.43 to 39.18% and 21.80 to 31.27% for germination treatments, respectively. Phytate content was significantly reduced from 23.59 to 32.40% for soaking treatment and 24.92 to 35.27% for germination treatments, respectively. Phytase enzymes will be activated during drying in equal form in all varieties. The results proved that the main distinct point is the change of phytase activity as well as specific activity during different treatment which showed no significant differences between the varieties used. The in vitro bioavailability of iron and zinc were significantly improved as a result of soaking and germination treatments.     

Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains

The phytase purple acid phosphatase (HvPAPhy_a) expressed during barley seed development was evaluated as transgene for overexpression in barley. The phytase was expressed constitutively driven by the cauliflower mosaic virus 35S‐promoter, and the phytase activity was measured in the mature grains, the green leaves and in the dry mature vegetative plant parts left after harvest of the grains. The T₂‐generation of HvPAPhy_a transformed barley showed phytase activity increases up to 19‐fold (29 000 phytase units (FTU) per kg in mature grains). Moreover, also in green leaves and mature dry straw, phytase activities were increased significantly by 110‐fold (52 000 FTU/kg) and 57‐fold (51 000 FTU/kg), respectively. The HvPAPhy_a‐transformed barley plants with high phytase activities possess triple potential utilities for the improvement of phosphate bioavailability. First of all, the utilization of the mature grains as feed to increase the release of bio‐available phosphate and minerals bound to the phytate of the grains; secondly, the utilization of the powdered straw either directly or phytase extracted hereof as a supplement to high phytate feed or food; and finally, the use of the stubble to be ploughed into the soil for mobilizing phytate‐bound phosphate for plant growth.     

Degradation of phytate in the gut of pigs ‐ pathway of gastrointestinal inositol phosphate hydrolysis and enzymes involved

The present study gives an overview on the whole mechanism of phytate degradation in the gut and the enzymes involved. Based on the similarity of the human and pigs gut, the study was carried out in pigs as model for humans. To differentiate between intrinsic feed phytases and endogenous phytases hydrolysing phytate in the gut, two diets, one high (control diet) and the other one very low in intrinsic feed phytases (phytase inactivated diet) were applied. In the chyme of stomach, small intestine and colon inositol phosphate isomers and activities of phytases and alkaline phosphatases were determined. In parallel total tract phytate degradation and apparent phosphorus digestibility were assessed. In the stomach chyme of pigs fed the control diet, comparable high phytase activity and strong phytate degradation were observed. The predominant phytate hydrolysis products were inositol phosphates, typically formed by plant phytases. For the phytase inactivated diet, comparable very low phytase activity and almost no phytate degradation in the stomach were determined. In the small intestine and colon, high activity of alkaline phosphatases and low activity of phytases were observed, irrespective of the diet fed. In the colon, stronger phytate degradation for the phytase inactivated diet than for the control diet was detected. Phytate degradation throughout the whole gut was nearly complete and very similar for both diets while the apparent availability of total phosphorus was significantly higher for the pigs fed the control diet than the phytase inactivated diet. The pathway of inositol phosphate hydrolysis in the gut has been elucidated.     

The relationship of alkaline phosphatase, CaATPase, and phytase

Alkaline phosphatase, highly purified from bovine intestinal mucosa, has significant hydrolytic activity against phytate and CaATP. Phytase and CaATPase activities require quite different assay conditions than those which are optimal for conventional alkaline phosphatase substrates such as 4-nitrophenyl phosphate. We have used affinity chromatography and antibody recognition to demonstrate that the phytase and CaATPase activities are not due to contaminating enzymes, but are intrinsic activities of intestinal alkaline phosphatase. All of the phytase and CaATPase activities present in crude extracts of bovine intestinal mucosa can be accounted for by alkaline phosphatase. Apparently neither phytase nor CaATPase exist in this tissue as independent enzymes. Specific substrates which require assay conditions quite different from the conventional 4-nitrophenyl phosphate substrate may account for the physiological function of "alkaline phosphatase."     

Improvement in cell‐bound phytase activity of Pichia anomala by permeabilization and applicability of permeabilized cells in soymilk dephytinization

Aims: Whole cell permeabilization of Pichia anomala to ameliorate the cell‐bound phytase activity and usability of permeabilized cells in dephytinization of soymilk. Methods and Results: The cells of P. anomala were subjected to permeabilization using the surfactant Triton X‐100 to overcome the permeability barrier and prepare whole cell biocatalysts with high phytase activity. The statistical approach, response surface methodology (RSM) was used to optimize the operating conditions for permeabilization. The treatment of cells with 5% Triton X‐100 for 30 min resulted in c. 15% enhancement in cell‐bound phytase activity. The shrinkage of protoplast was observed, although cell viability and phytase stability were not significantly altered. The free as well as immobilized permeabilized cells hydrolysed soymilk phytate, and the latter could be reused over four consecutive cycles. Conclusions: Whole cell permeabilization of P. anomala using Triton X‐100 led to enhancement in cell‐bound phytase activity. The viability and integrity of yeast cells were not significantly affected because of permeabilization. The permeabilized P. anomala cells effectively dephytinized soymilk, and the permeabilized cells immobilized in alginate could be reused because of sustained phytase activity. Significance and Impact of the Study: This is the first report on the use of permeabilized yeast cells for mitigating phytate content of soymilk. Alginate entrapment of permeabilized P. anomala allows reuse of cells for soymilk dephytinization, thus suggesting a potential application in food industry.     

Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states

Phytases hydrolyze phytic acid to less phosphorylated myo-inositol derivatives and inorganic phosphate. A thermostable phytase is of great value in applications for improving phosphate and metal ion availability in animal feed, and thereby reducing phosphate pollution to the environment. Here, we report a new folding architecture of a six-bladed propeller for phosphatase activity revealed by the 2.1 A crystal structures of a novel, thermostable phytase determined in both the partially and fully Ca2+-loaded states. Binding of two calcium ions to high-affinity calcium binding sites results in a dramatic increase in thermostability (by as much as approximately 30 degrees C in melting temperature) by joining loop segments remote in the amino acid sequence. Binding of three additional calcium ions to low-affinity calcium binding sites at the top of the molecule turns on the catalytic activity of the enzyme by converting the highly negatively charged cleft into a favorable environment for the binding of phytate.     

A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100°C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20–30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed–batch cultivation, glucose concentration was maintained at 2–3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.     

Directed evolution of a highly active Yersinia mollaretii phytase

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073?U/mg which is ～10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ～8,400 clones) a phytase variant with ～20% improved thermostability (58°C for 20?min; residual activity wild type ～34%; variant ～53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993?U/mg).     

Biochemical properties and substrate specificities of alkaline and histidine acid phytases

Phytases are a special class of phosphatase that catalyze the sequential hydrolysis of phytate to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are added to animal feedstuff to reduce phosphate pollution in the environment, since monogastric animals such as pigs, poultry, and fish are unable to metabolize phytate. Based on biochemical properties and amino acid sequence alignment, phytases can be categorized into two major classes, the histidine acid phytases and the alkaline phytases. The histidine acid phosphatase class shows broad substrate specificity and hydrolyzes metal-free phytate at the acidic pH range and produces myo-inositol monophosphate as the final product. In contrast, the alkaline phytase class exhibits strict substrate specificity for the calcium–phytate complex and produces myo-inositol trisphosphate as the final product. This review describes recent findings that present novel viewpoints concerning the molecular basis of phytase classification.     

Effect of calcium,copper, and zinc levels in a rapeseed meal diet on mineral and trace element utilization in the rat

Mineral and trace element interactions were studied in a balance trial with rats. Calcium, copper, and zinc were supplied to a rapeseed meal diet in a factorial design. Animals were fedad libitum, and absorption, excretion, and retention of the elements were evaluated either as fractions of total intake or in relation to nitrogen retention to account for differences in food intake and lean body mass increment. The intrinsic content of minerals and trace elements was sufficient to support growth at a rate that could be expected from the rapeseed protein quality. However, when calcium was included in the diet, the intrinsic dietary level of zinc appeared to be limiting, despite the fact that the zinc level was twice the recommended level. Additional zinc supply reversed growth impairment. This calcium-zinc interaction is believed to be owing to the formation of phytate complexes. Calcium addition influenced the calcium, phosphorus, magnesium, zinc, and iron—but not the copper—balances. The addition of calcium reduced the availability of the intrinsic zinc, whereas no effect was seen in the zinc-fortified groups. The availability of intrinsic copper was in a similar way significantly impaired by addition of dietary zinc, where-as copper-supplied groups were unaffected by zinc addition. Intrinsic iron availability was also dependent upon zinc addition, although in a more ambigouus way. Thus, addition of extrinsic minerals to a diet high in phytate can result in significant impairments of growth and mineral utilization.     

Microbial phytase addition resulted in a greater increase in phosphorus digestibility in dry-fed compared with liquid-fed non-heat-treated wheat–barley–maize diets for pigs

The objective was to evaluate the effect of microbial phytase (1250 FTU/kg diet with 88% dry matter (DM)) on apparent total tract digestibility (ATTD) of phosphorus (P) in pigs fed a dry or soaked diet. Twenty-four pigs (65±3 kg) from six litters were used. Pigs were housed in metabolism crates and fed one of four diets for 12 days; 5 days for adaptation and 7 days for total, but separate collection of feces and urine. The basal diet was composed of wheat, barley, maize, soybean meal and no mineral phosphate. Dietary treatments were: basal dry-fed diet (BDD), BDD with microbial phytase (BDD+phy), BDD soaked for 24 h at 20°C before feeding (BDS) and BDS with microbial phytase (BDS+phy). Supplementation of microbial phytase increased ATTD of DM and crude protein (N×6.25) by 2 and 3 percentage units (P<0.0001; P<0.001), respectively. The ATTD of P was affected by the interaction between microbial phytase and soaking (P=0.02). This was due to a greater increase in ATTD of P by soaking of the diet containing solely plant phytase compared with the diet supplemented with microbial phytase: 35%, 65%, 44% and 68% for BDD, BDD+phy, BSD and BSD+phy, respectively. As such, supplementation of microbial phytase increased ATTD of P in the dry-fed diet, but not in the soaked diet. The higher ATTD of P for BDS compared with BDD resulted from the degradation of 54% of the phytate in BDS by wheat and barley phytases during soaking. On the other hand, soaking of BDS+phy did not increase ATTD of P significantly compared with BDD+phy despite that 76% of the phytate in BDS+phy was degraded before feeding. In conclusion, soaking of BDS containing solely plant phytase provided a great potential for increasing ATTD of P. However, this potential was not present when microbial phytase (1250 FTU/kg diet) was supplemented, most likely because soaking of BDS+phy for 24 h at 20°C did not result in a complete degradation of phytate before feeding.     

Effects of nutritional deficiencies during neonatal and post-weaning period on rat intestinal phytase and phosphatase activities

Studies were made of the effects of pre- and post-weaning undernutrition and/or protein deficiency on intestinal phytase and phosphatase activities in albino rats and reversibility of the same by subsequent dietary rehabilitation. Neonatal undernutrition induced by rearing the pups in litters of 16 caused a marked decrease in alkaline phytase activity (as compared to those reared in litters of 8), while acid phytase activity decreased to a lesser extent and acid and alkaline phosphatase activities did not change. When neonatally undernourished rats were subsequently continued on a 4 or a 20% protein diet in restricted amounts (2.5 g/day) for 6 weeks the decreases in the alkaline phytase activity but not in that of acid phytase were further aggravated. Acid and alkaline phosphatases were not influenced by these treatments either. On dietary rehabilitation of these rats for subsequent 6 weeks on a 20% protein diet (ad libitum) acid and alkaline phytase activities of intestine recovered partially. These studies indicate the importance of alkaline phytase activity as a marker of intestinal maturation and is also suggestive of interrelationships between nutrition, intestinal development and its alkaline phytase activity.     

Isolation, Characterization, Molecular Gene Cloning, and Sequencing of a Novel Phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55°C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.     

Phytase produced on citric byproducts: purification and characterization

Citric pulp is an agro-industrial residue from the citrus processing industry with low inorganic phosphorus content applied in animal feed. A new bioprocess was developed to produce and purify a new phytase generated on citric pulp fermentation by Aspergillus niger FS3. The phytase was purified by cationic-exchange, anionic-exchange chromatography and chromatofocusing steps. From SDS–PAGE analysis, the molecular weight of the purified phytase was calculated to be 108 kDa. The phytase had an optimum pH of 5.0–5.5 and an optimum temperature of 60°C. The phytase displayed high affinity for phytate, and the K _m was 0.52 mM. The purified phytase was sufficiently able to withstand pelleting temperatures, retaining sufficiently high phytate-degrading activity.     

Recombinant,Rice-Produced Yeast Phytase Shows the Ability to Hydrolyze Phytate Derived from Seed-Based Feed,and Extreme Stability during Ensilage Treatment

When fresh rice leaves producing yeast Schwanniomyces occidentalis phytase were grounded and mixed with the whole extract of seed-based feed for pigs, the release of orthophosphate increased significantly. More specifically, phytate, a major source of phosphorus in the seeds, was hydrolyzed by heterologous phytase. Moreover, when transgenic rice plants were ensiled for up to 12 weeks, no decrease in the phytase activity of the heterologous enzyme was observed. This result strongly suggests that transgenic rice plants producing yeast phytase can be stored as silage without any loss of enzyme activity until usage as a feed additive.     

Production of feed enzymes (phytase and plant cell wall hydrolyzing enzymes) by<Emphasis Type="Italic">Mucor indicus</Emphasis> MTCC 6333: Purification and characterization of phytase

The production of phytase and associated feed enzymes (phosphatase, xylanase, CMCase, alpha-amylase and beta-glucosidase) was determined in a thermotolerant fungus Mucor indicus MTCC 6333, isolated from composting soil. Solid-substrate culturing on wheat bran and optimizing other culture conditions (C and N sources, level of N, temperature, pH, culture age, inoculum level), increased the yield of phytase from 266 +/- 0.2 to 513 +/- 0.4 nkat/g substrate dry mass. The culture extract also contained 112, 194, 171, 396, and 333 nkat/g substrate of phosphatase, xylanase, CMCase, beta-glucosidase and alpha-amylase activities, respectively. Simple 2-step purification employing anion exchange and gel filtration chromatography resulted in 21.9-fold purified phytase. The optimum pH and temperature were pH 6.0 and 70 degrees C, respectively. The phytase was thermostable under acidic conditions, showing 82% residual activity after exposure to 60 degrees C at pH 3.0 and 5.0 for 2 h, and displayed broad substrate specificity. The Km was 200 nmol/L and v(lim) of 113 nmol/s per mg protein with dodecasodium phytate as substrate. In vitro feed trial with feed enzyme resulted in the release of 1.68 g inorganic P/kg of feed after 6 h of incubation at 37 degrees C.     

<Emphasis Type="Italic">Pichia anomala</Emphasis> in grain biopreservation

Cereal grain is a major component of food and feed in large parts of the world. The microbial flora on cereal grains may interfere with hygiene and storage stability, palatability and bioavailability of minerals and proteins may depend on the composition of the microbial population. Therefore, it is of primary interest to control the microbial species present on cereal grain. Inoculation of the biocontrol yeast Pichia anomala to cereal feed grain improved feed hygiene by reduction of moulds and Enterobacteriaceae, and enhanced the nutritional value by increasing the protein content and reducing the concentration of the antinutritional compound phytate. P. anomala strains showed a high phytase activity, for some strains also considerable extracellular phytase activity was observed. A certain maximum in biomass concentration was never exceeded indicating cell density induced growth inhibition of P. anomala.     

Tissues and organs as indicators of intestinal absorption of minerals and trace elements,evaluated in rats

Tissue and organ deposition and blood parameters were evaluated as indices of mineral and trace element absorption in rats. The absorption of elements was quantified in relation to nitrogen retention, i.e., considering the weight gain and new tissue synthesis. A rapeseed meal diet was supplied with three levels of calcium, two levels of zinc, and two levels of copper in a factorial design. In general, an increase in dietary mineral content increased the relative absorption, which in turn, increased the tissue deposition progressively. Striated muscle, however, did not respond to either an increased calcium or zinc supply. Furthermore, an increased calcium absorption caused a depression of the fractional phosphorus and magnesium content of femur bones. The copper content of the kidneys and the heart muscle was directly proportional to the amount of absorbed zinc and iron, respectively. The iron content of tissues was, in general, inversely proportional to zinc absorption and showed a tendency to be directly proportional to copper absorption. The zinc level in tissues was, in a similar way, inversely correlated to measured calcium absorption. In conclusion, interactions between elements do not only affect the intestinal element absorption, but also the distribution of already absorbed elements in tissues and organs.     

Purification and Characterization of a Bacterial Phytase Whose Properties Make it Exceptionally Useful as a Feed Supplement

A periplasmatic phytase from a bacterium isolated from Malaysian waste water was purified about 173-fold to apparent homogeneity with a recovery of 10% referred to the phytase activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 65°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K _M = 0.15 mmol/l and k _cat = 1164 s⁻¹ at pH 4.5 and 37°C. The purified enzyme was shown to be highly specific. Among the phosphorylated compounds tested, phytate was the only one which was significantly hydrolysed. Some properties such as considerable activity below pH 3.0, thermal stability and resistance to pepsin make the enzyme attractive for an application as a feed supplement.