Characterization of recombinant phytase of Klebsiella sp. and the influence of novel 3-phytase on mineral solubility in broiler diets under an in vitro digestion assay.

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.

Effects of phytase enzyme supplementation on growth performance, intestinal morphology and metabolism in Nile tilapia (Oreochromis niloticus).

Phytase is crucial in enhancing the bioavailability and release of phosphorus and other nutrients bound to phytic acid, making them more bioavailable for animal absorption. This study was carried out to inspect the effect of supplementing low phosphorus (P) diet with di-calcium phosphate (DCP) and liquid phytase enzyme (LP), which contains 1500 FTU/kg, on growth performance, intestinal morphometry, proximate body chemical composition, blood profile, immunity status, liver mitochondrial enzyme activities, the expression response and economic returns of Nile tilapia (Oreochromis niloticus). Three triplicate groups of fish (initial weight 5.405 ± 0.045 g, N = 90) were fed on three different diets for 90 days. The first was a control diet with zero DCP; the second was a control diet supplemented with 0.71% DCP; the third was a control diet supplemented with 0.03% LP. The groups were designated as CG, DCP and LP, respectively. Results showed that LP induced considerable improvements (p < 0.05) in FBW, body weight gain, weight gain rate, specific growth rate, HIS, viscero-somatic index, spleen-somatic index, feed conversion ratio, blood parameters and the histomorphometry assessment of intestinal villi absorptive capacity, compared with the other groups. Also, whole-body protein and lipid contents pointedly (p < 0.05) increased by LP, compared with the DCP group. A positive response (p < 0.05) to the phytase enzyme was noted in complexes I, III and IV of the mitochondrial liver complex enzyme activity. Likewise, the relative gene expression levels of (GHr-1, IGF-1, FAS and LPL) were notably (p < 0.05) upregulated by phytase enzyme, associated with DCP and control groups. Further, phytase recorded the highest total return and profit percentage. It can be concluded that Nile tilapia benefits from using phytase enzyme 1500 FTU/kg at 0.03% without adding DCP in terms of good performance and profits.

Impact of Fermentation, Autoclaving and Phytase Treatment on the Antioxidant Properties and Quality of Teff Cookies.

Research background: Teff [Eragrostis tef (Zucc.) Trotter] is an underutilised cereal crop grown mainly in Ethiopia and Eritrea. It is an excellent source of dietary fibre, vitamins, minerals and bioactive compounds. However, it also contains a high amount of phytic acid, which is an antinutrient and reduces the bioavailability of minerals and proteins. To improve the nutritional quality of teff, the phytic acid content should be reduced by an effective dephytinisation method. Experimental approach: In this study, various dephytinisation methods (fermentation, autoclaving and phytase treatment) were used to dephytinise teff flour. Undephytinised and dephytinised teff flour was mixed into wheat flour (0-40 %) to improve the functional properties of cookies. Twenty different cookie formulations were prepared according to 4x5x2 factorial design. The physical, chemical, nutritional and sensory properties of the cookies were investigated. Results and conclusions: Among the dephytinisation methods, fermentation produced the most effective reduction in phytic acid mass fraction (181 mg/100 g), followed by phytase treatment (198 mg/100 g). The protein, fat, Fe and Zn content and antioxidant activity of cookies enriched with dephytinised teff flour were comparable to cookies fortified with undephytinised teff flour. Moreover, the dephytinised teff cookies had lower phytic acid mass fractions. The cookies containing 40 % teff flour had higher antioxidant activity and nutritional quality than the control wheat cookies. The use of dephytinised teff flour reduced the spread ratio and the a* and b* values of cookies compared to undephytinised flour. Cookies containing fermented and phytase-treated teff flour had a harder texture than cookies containing undephytinised flour. In addition, as the amount of teff flour increased, the spread ratio values of cookies gradually incrased while their hardness decreased. Overall acceptability scores of cookies containing 10-20 % teff flour were similar to the control. Novelty and scientific contribution: To the best of our knowledge, this is the first study to determine the quality of cookies containing dephytinised teff flour. The data highlight the potential of dephytinised (especially autoclaved and phytase-treated) teff flour (up to 20 %) as a functional ingredient to enrich the mineral content and antioxidant capacity of foods. Furthermore, this study shows that fermentation, autoclaving and phytase treatment can be used to improve the nutritional quality of grains.

Effect of phytase enzyme on growth performance, serum biochemical alteration, immune response and gene expression in Nile tilapia.

The present study aimed to investigate the effect of low phosphorus diet with or without different levels of phytase enzyme supplementation on growth performance, body composition, nutrient retention efficiency, gene expression, and health status of A. hydrophila challenged fish. A total of 240 monosex males of Nile tilapia (Oreochromis niloticus) with an average body weight of 23.19 ±â€¯0.15 g/fish were used. Fish were randomly chosen and divided into 4 equal groups (60 fish per group), with 3 subgroups containing 20 fish as a replicate. Group 1, was fed on a diet containing 100% P, group 2, was fed on a diet containing 50% P, group 3 and 4, were fed on low P with 500 or 1000 units of phytase/Kg respectively. It was observed that the 50% phosphorus diet significantly reduced body weight, feed intake, feed conversion ratio (FCR), and protein efficiency ratio (PER) compared to Nile tilapia fish fed on the diet containing 100% phosphorus. In contrast, fish fed on the diet containing 50% phosphorus supplemented by 500 or 1000 phytase units/kg significantly (P ≤ 0.05) increased final body weight (FBW), total body gain (TBG), average daily gain (ADG), and weight gain compared to Nile tilapia fed on the same diet or fed on the diet containing normal phosphorus without phytase supplementation. Different phosphorus and phytase supplementation levels had no significant effect on serum total protein, albumin, and globulin concentrations, meanwhile, phytase supplementation increased serum calcium and phosphorus levels. Nile tilapia fed on phytase supplementation had an increase in body protein, lipid content, and nutrient utilization efficiency compared to Nile tilapia fed on the diet containing 100% phosphorus. Nile tilapia fed on low dietary phosphorus showed an increase in mortality after infection and a decrease in phagocytosis and neutrophil compared to fish fed on normal phosphorus. Phytase supplementation, made immune response parameters return to its normal values and the pathological lesions of liver, spleen, stomach, and intestine were reduced. Moreover, normal phosphorus significantly up-regulated lipoprotein lipase (LPL) mRNA expression and down-regulated fatty acid synthase (FAS) mRNA in Nile tilapia's liver while low phosphorus with or without phytase supplementation reduced LPL expression and relatively up-regulated FAS.

Effects of Dietary Supplementation of Arginine-Silicate- Inositol and Phytase Complex on Performance and Blood Biochemical Traits of Laying Hens.

Arginine silicate inositol complex (ASI; Arg = 49.47%, silicone = 8.2%, inositol = 25%) is a novel, bioavailable source of Si and Arg and may offer potential benefits for laying hens' performance. The aim of this study was to evaluate the effect of Arginine-Silicate and inositol/phytase on the performance of laying hens. A total of 90 laying hens, 25 weeks old, were randomly assigned to 6 treatments with 3 replicates (5 birds per replicate). The treatments were as follows: 1ST treatment PC: positive Control group (basal diet without additives (, 2nd treatment: basal diet +1000 mg/kg arginine-silicate complex (49.5±8.2 % respectively), 3d treatment: basal diet +1000 mg/kg arginine-silicate- inositol (ASI) complex (49.5, 8.2 , 25 % respectively) , 4th treatment: T 2 +500 FTU/kg , 5th treatment: T2 +1000 FTU/kg and 6th treatment: T2+2000 FTU/kg . Results indicate a significant increase (P<0.05) in hen house production (H.H. pro.%) of T5 (95.06 %)compared with T1(91.67%) and no significant differences between T2, T3, T4, T6 (91.84, 93.21, 93.46, 92.98%) and compared with T1 and T5. were no significant difference observed in average egg weight and egg mass between the experimental treatments all over the period. Daily feed intake (DFI) significantly decreased (P<0.05) with supplementing diets with deferent levels of phytase with arginine-silicate mixture T4, T5, andT6 (113.56،113.06، 112.10 g) compared with T1 (114.34 g ) which has no significant differences compared with T2 and T3 (113.96, 113.92 g). Phytase supplementation significantly (P<0.05) improved FCR g feed/egg in T5 (119.02) compared with T1 and T2 (124.89, 124.32), while no significant differences between T3.T4.T6 treatments (122.39, 121.80, 120.69) respectively and compared with other treatments. The experimental treatments observed no significant difference in g feed/ g egg.

Fluorometric detection of phytase enzyme activity and phosphate ion based on gelatin supported silver nanoclusters.

Phytase is the commercial enzyme for bioconversion of phytate substrate to digestible phosphate ions. Recently silver nanoclusters (AgNCs) have received great attention as the optical transducer nanoparticles in biosensors structure. The novel detection platform was developed to detect the phytase enzyme activity and phosphate ions based on fluorescence quenching of AgNCs. The AgNCs were synthesized through gelatin supported reaction and characterized by TEM, FTIR and XRD analysis. The hydrolytic effect of phytase enzyme and subsequent phosphate release led to suppression of AgNCs fluorescence. The linear range was observed for enzyme in the range of 0.5-5 U/mL with the detection limit of 0.2 U/mL. Also, the same fluorescence quenching effect was observed in the presence of phosphate ion in the linear range of 1 to 16 µM with a detection limit of 0.5 µM. The proposed mechanism showed effectiveness of detection strategy for detection of phytase enzyme and phosphate ion.

Rice bran nanofiber composites for stabilization of phytase.

This study explores the potential application of rice bran (agro waste) to nano-encapsulate phytase, which is a thermally unstable biologically active enzyme. Rice bran was converted to nanofibers (20-50 nm in diameter) using electrospinning. After optimizing the pH, viscosity, voltage and the distance between electrodes for electrospinning, phytase enzyme was encapsulated and the fibers were cross-linked using sodium tripolyphosphate. Thermal stability of phytase enzyme was improved by 90 °C when they are encapsulated and cross-linked with sodium tripolyphosphate. The activity of the phytase enzyme was monitored at different temperatures. The activity of the pure enzyme was lost at 80 °C while the enzyme encapsulated into nanofibers demonstrated the activity up to 170 °C. This study opens up many opportunities for nanotechnology value addition to many waste materials and also to improve the properties of a range of biomaterials through a sustainable approach.

Effects of dietary supplementation of organic acids and phytase on performance and intestinal histomorphology of broilers.

The present experiment was conducted to evaluate the effects of organic acids and phytase enzyme supplementation on performance and intestinal histomorphology of broilers. The experiment was done in a factorial arrangement 2 × 2 × 2 based on completely randomized design with eight treatments, five replicates with 12 chicks in each until 42 days of age. Diets included natural vinegar (0 and 2%), citric acid (CA; 0.00 and 1.00%) and phytase enzyme (PHY; 0.00 and 500 FTU phytase per kg of feed). One bird from each treatment replicate was randomly selected and slaughtered to evaluate the small intestinal morphology on 42 days of age. Analysis of results showed that vinegar increased feed consumption and body weight gain in total experimental period (p ˂ 0.05), while CA significantly decreased feed consumption on 0-14 days of age (p ˂ 0.05). No effect was observed on performance in interaction of organic acids together and with PHY group (p > 0.05). In duodenum CA increased the villus height and width (p ˂ 0.05) and PHY enzyme increased villus width (p ˂ 0.05) and decreased crypt depth (p ˂ 0.05). On the other hand, CA along with PHY significantly decreased crypt depth (p ˂ 0.05). In jejunum PHY alone and in combination with vinegar increased the goblet cells numbers (p ˂ 0.05), whereas vinegar significantly increased the goblet cells numbers in ileum (p ˂ 0.05). The muscular thickness in duodenum, jejunum, and ileum was not affected among different treatment groups. The results showed that supplementation of organic acids and phytase together in this experiment, with no negative effects on each other, improved their effects on some parameters.

Radioisótopo e modelagem matemática na avaliação da suplementação de fitase em dietas para suínos em crescimento / Radioisotope and mathematical modeling in the assessment of supplementation of Phytase in diets for growing pigs

O objetivo deste trabalho foi avaliar os efeitos da inclusão de níveis crescentes de enzima fitase em dietas para suínos em fase de crescimento, por meio do uso de modelagem matemática. Foram utilizados dados de 20 leitões mestiços, machos castrados, pesando em média, 26,8kg. Os animais ficaram alojados em gaiolas metabólicas individuais para a coleta de fezes e urina, onde permaneceram por um período de 17 dias. Utilizou-se o delineamento experimental de blocos ao acaso, com cinco tratamentos e quatro repetições. A dieta experimental fornecida aos leitões foi à base de milho e farelo de soja, suplementada com cinco níveis crescentes de enzima fitase (0, 250, 500, 750 e 1000UF/kg), correspondendo a 0; 0,01; 0,02; 0,03 e 0,04%, respectivamente. As variáveis avaliadas foram: ingestão, excreção, fluxo e refluxo do P nos compartimentos (trato digestivo, corrente sanguínea, tecidos moles e ossos). A enzima fitase não interferiu no P consumido (P>0,05 (F10), no P excretado na urina (F02), no fluxo e refluxo do P nos ossos (F32 e F23) e nos tecidos moles (F42 e F24), entretanto observou-se redução no P excretado nas fezes (F01) em 8,92%; 26,76%; 22,53% e 28,64% para os níveis 0, 250, 500, 750 e 1000UF/kg, respectivamente, e efeito linear positivo para o P endógeno (F12). Pode-se utilizar dietas à base de milho e farelo de soja com 50% de P por fosfato bicálcico, adicionando-se 250UF/kg de dieta para suínos em crescimento, o que reduz em 27% as excreções de P nas fezes.

The purpose of this study was to evaluate the effects of including increasing levels of phytase enzyme in pig diets for growing pigs, using the mathematical model. Data from 20 crossbred male piglets, castrated and weighing 26.80 kg on average was used. The animals were housed in individual metabolic cages to collect feces and urine in a 17 day period. A randomized block experimental design containing five treatments and four repetitions was used. The experimental diet provided to piglets contained corn and soybean and was supplemented with five increasing levels of phytase enzyme (0, 250, 500, 750 and 1000 UF/kg), corresponding to 0.00 %, 0.01 %, 0.02 %, 0.03 % and 0.04 % respectively. The variables evaluated were: intake, excretion, output flow of P in the digestive tract, bloodstream, bones and soft tissues. The phytase enzyme did not affect the P intake (P>0.05 (F 10), the P excreted in urine (F02) and the output flow of P in the bones (F32 e F23) and soft tissue (F42 e F24) , however, there was a reduction in P excreted in feces (F01) of 8.92 %, 26.76 %, 22.53 % and 28.64 % to the levels 0, 250, 500, 750 e 1000UF/kg, respectively and showed a positive linear effect (P<0.08) for the endogenous P (F12) . Corn and soybean meal based diets can be used with 50% of P by dicalcium phosphate, adding 250UF/kg diet for growing pigs, and may cause a reduction of 27 % of P excretion in feces.

Modelos matemáticos para o estudo do fluxo biológico do fósforo em suínos alimentados com dietas suplementadas com níveis crescentes de fitase / Mathematical models for the study of the biological flow of phosphorus in pigs fed with diets containing increasing levels of phytase

A pesquisa foi realizada para avaliar o fluxo biológico do fósforo entre os compartimentos, fisiológicos ou anatômicos, de suínos mantidos em dietas que continham níveis crescentes de fitase, usando o P-32 como traçador. O delineamento experimental foi inteiramente ao acaso, sendo os tratamentos constituídos por níveis de enzima fitase, 253, 759, 1265 e 1748 UF/kg, nas dietas. As variáveis avaliadas foram: ingestão, excreção, atividades específicas e fluxo de P nos compartimentos (trato digestivo, corrente sanguínea, tecidos moles e ossos). A enzima fitase não interferiu nas excreções fecais e urinárias, nas atividades específicas, na incorporação e na reabsorção de P nos ossos e nos tecidos moles e no fluxo bidirecional do trato digestivo e na corrente sanguínea, mas afetou linearmente a absorção e a retenção nos ossos. As respostas mais evidentes dos efeitos da adição da fitase são observadas nos níveis mais baixos.

The biological flow of P was evaluated among the physiologic or the anatomical compartments of pigs fed diets with increasing phytase levels, using P-32 as tracer. The experimental design was completely randomized. Treatments consisted of phytase levels in diets (253, 759, 1265, and 1748UF/kg). The evaluated variables were: feed intake, excretion, specific activities and flow of P in the compartments (gut, blood, bone, and soft tissus). Phytase level did not interfere with fecal and urinary excretion, specific activities, incorporation and resorption of bone, and soft tissue P and in the bidirectional flow of gut and blood, but linearly affected P absorption and bone P retention. The best response was observed with the lowest phytase levels.