In-vitro and in-vivo dephosphorylation of rapeseed meal by means of phytate-degrading enzymes derived from Aspergillus Niger

A meal of ‘double low’ rapeseed (var ‘Jantar’) was subjected to phytate hydrolysis using enzyme preparations derived from a mycelium of Aspergillus niger which contained phytase (EC 3.1.3.8) and acid phosphatase (EC 3.1.3.2) activities. The complete conversion of myo-inositol hexa- and pentaphosphates present in rapeseed meal to lower phosphate esters of myo-inositol was accomplished at 40°C, a pH value of 4.5, phytase dosage (in phytase units (PhytU)) 0.1 PhytU g^?1 accompanied by acid phosphatase activity 37.1 units g^?1, in 1 h. Under these conditions, complete dephosphorylation was observed in 4 h. Decreasing the pH value to 3.0 caused a rise in the amount of inorganic phosphorus released, while increasing to 5.5 resulted in substantial reduction in the reaction rate. Purification of phytase to a specific activity 0.375 PhytU mg^?1 of protein exhibited a negative influence upon the yield of rapeseed dephosphorylation. The substitution of calcium phosphate for a preparation of phytase in feed containing rapeseed meal did not cause significant differences in the body weight gain or in tibia mineralisation of broilers (Gains galus, ‘Astra B’).     

Hydrolysis of galactooligosaccharides by commercial preparations of α-galactosidase and β-fruetofuranosidase: Potential for use as dietary additives

In vitro and in vivo studies were conducted with six commercial enzyme preparations (SP249, Energex, Rohament CW, Novozyme 230 and crude α -galactosidase) to determine their effectiveness in hydrolysing galactooligosaccharides from soya bean and canola meal in the gastrointestinal tract of poultry. The use of the enzyme invertase to enhance galactoside hydrolysis was also studied. A wide range of α -galactosidase activity was observed in vitro, with crude α-galactosidase from Mortirella vinacea and Novozyme 230 preparation showing the highest activity values of 4.3 and 1.5 nkat mg^?1, respectively. All preparations with the exception of crude α-galactosidase showed invertase activity which is known to convert raffinose and stachyose to the corresponding di-and trisaccharide, melibiose and manninotriose. Although the activity of invertase was highest on sucrose, the Novozyme 230 preparation showed activity values of 4.2 and 2.3 nkat mg^?1 toward raffinose and stachyose substrates, respectively. De novo synthesis of raffinose was observed when soya bean meal, canola meal or pure sucrose and galactose were incubated with certain enzyme preparations (ie Energex). In general, preparations possessing hydrolytic activity towards galactooligosaccharides showed very little synthesis of raffinose while preparations capable of generating raffinose were very weak in the hydrolysis of galactooligosaccharides. The best result in terms of galactooligosaccharide in vitro hydrolysis of canola and soya bean meal was obtained with a combination of α-galactosidase and invertase. In the in vivo study with caecectomised hens, hydrolysis of galactooligosaccharides averaged 88% when crude α-galactosidase (2 g kg^?1) and invertase (1 g kg^?1) were added to laying, hen diet containing 200 g soya bean meal per kilogram. A problem identified in the current study was that minerals such as calcium phosphate and calcium carbonate common in poultry diets inhibit the hydrolysis activity of α-galactosidase, indicating that high levels of activity would be required to yield a response in practical poultry feeding.     

Peniophora lycii phytase is stabile and degrades phytate and solubilises minerals in vitro during simulation of gastrointestinal digestion in the pig

BACKGROUND: Microbial phytases (EC 3.1.3) are widely used in diets for monogastric animals to hydrolyse phytate present in the feed and thereby increase phosphorus and mineral availability. Previous work has shown that phytate solubility is strongly affected by calcium in the feed and by pH in the gastrointestinal (GI) tract, which may have an effect on phytase efficacy. An in vitro model simulating the GI tract of pigs was used to study the survival of Peniophora lycii phytase and the effect of the phytase on phytate degradation, inositol phosphate formation and mineral solubilisation during in vitro digestion of a 30:70 soybean meal/maize meal blend with different calcium levels. RESULTS: The phytase retained 76 and 80% of its initial activity throughout the gastric in vitro digestion. Total phytate hydrolysis by P. lycii phytase was in the same range at total calcium levels of 1.2 and 6.2 mg g^?1 dry matter (DM), despite very large differences in phytate solubility at these calcium levels. However, at 11.2 and 21.2 mg Ca g^?1 DM, phytate hydrolysis was significantly lower. The amount of soluble mineral was generally increased by P. lycii phytase. CONCLUSION: Stability of P. lycii phytase during gastric digestion was not found to be critical for phytate hydrolysis. Furthermore, original phytate solubility was not an absolute requirement for phytate degradation; phytate solubility seemed to be in a steady state, allowing insoluble phytate to solubilise as soluble phytate was degraded. This is new and interesting knowledge that adds to the current understanding of phytate–phytase interaction. Copyright © 2007 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF A PHYTASE FROM SPELT

Four soluble phytases were identified in germinating spelt. Although numerous purification strategies were applied, none of the four phytases could be purified to homogeneity. The purest phytase preparation, called D21, contained a phytase (major component) and an acid phosphatase (APH) (minor component). The phytase behaves like a monomeric protein of a molecular mass of about 68 kDa and shows a broad substrate specificity. Optimal pH for degradation of phytate was 6.0 and the optimal temperature 45C. Kinetic parameters for the hydrolysis of Na-phytate were K_M 400 μmoll^?1 and k_cat 368s^?1 at pH 6.0. The spelt phytase D21 degrades phytate stepwise.     

The nutritional quality of two types of fish silage for broiler chickens

Fish waste was ensiled either by acidification with formic acid or by fermentation with a bacterial starter culture and molasses. The resulting liquids were mixed with wheat bran (85:15 w/w liquid: bran) and dried (70°C) to produce acid silage meal (ASM) and fermented silage meal (FSM). ASM and FSM were incorporated into wheat-based diets at 25, 50 and 100 g kg^?1 at the expense of soya bean meal. There were two control diets, one which contained soya bean meal as the predominant protein supplement and a second in which fish meal (50 g kg^?1) was added at the expense of some of the soya bean meal. Starter diets (13.25 MJ ME kg^?1, 12 g kg^?1 lysine) were fed from 1-day-old to 21-days-old and finisher diets (13.25 MJ ME kg^?1, 9.5 g kg^?1 lysine) were fed from 22 to 42-days-old to six replicates each of five birds. Birds were reared in raised-wire cages, and feed intake, liveweight and mortality were recorded. There were no significant effects of dietary inclusion of either ASM and FSM on the performance of broiler chickens relative to those fed on control diets. FSM contained less crude protein and amino acids than ASM. The recovery of amino acids relative to the total crude protein content from FSM was only 78.7%, presumably as a result of formation of Maillard reaction products during drying.     

Partial characterisation of a new barley amylase

A new amylolytic enzyme found in barley (Hordeum vulgare) grain was partially characterised with respect to physicochemical properties and enzymatic activity. The enzyme preparation showed one antigenically homogeneous amylolytic band. Isoelectric focusing resolved the new amylase into two components, one isoelectric at pH 4.5, the other at pH 3.0. During focusing the original activity of the new amylase decreased by 80%. The purified preparation was inactivated by pH-values below 4.5 and above 9.0 and also by temperatures above + 40°C for 1 h. The new amylase splits the 1,4-α-glycosidic linkages, clearly by endo-attack, of starch, amylopectin, amylose and β-limit dextrin optimally at pH 6.5 at +40°C giving K_m-values 8.9 × 10^?3, 4.4 × 10^?3, 6.6 × 10^?3 and 1.7 × 10^?3 g/ml, respectively. The hydrolysis products from β-limit dextrin were 24% glucose and 46% maltose in the total digest. Mercuric chloride, pCMB,^a EDTA^a and TRIS^a have no noticeable effect on the new amylase, indicating that it is stable under conditions where the other amylolytic enzymes are deactivated. The new amylase seems to be a hydrolase acting on o-glycosyl compounds, EC 3.2.1., but could not be identified with any of the amylolytic enzymes of vegetable origin studied previously.     

Original article: Thermal pretreatment and chemical modifications as a means to alter hydrolytic characteristics and prevent bitterness in hydrolysates of fishery by‐catch (Decapterus maruadsi) protein

In this study, fishery by‐catch protein (Decapterus maruadsi) was first thermal treated (60–100 °C), phosphorylated (0.5–4.0 g g^?1 protein) or succinylated (0.05–0.3 g g^?1 protein) before hydrolysis to prevent the bitterness of the resulting hydrolysate. Hydrolysis was performed at 50 °C, 10 h, pH 8.0 by Alcalase. Results showed that thermal treatment upon 80 °C, succinylation and phosphorylation resulted in significant decrease in amino acid content and increase in larger peptide content (>1450 Da). Succinylation did not reduce the bitterness as a result of higher content of small peptides (<550 Da), which was found to be correlated positively with the bitterness of hydrolysates. However, thermal treatment, especially phosphorylation, could reduce the bitterness to discernible level, indicating that phosphorylation before hydrolysis could be a potential means to prevent the evolution of bitter flavour which restricts the practical uses of the hydrolysates.     

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     

Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humates

Alkaline phosphatase (EC 3.1.3.1) extracted from Escherichia coli ATCC27257 was immobilised by co‐flocculation with soil humates in the presence of Ca²⁺. The effects of time, temperature, pH and concentration of enzyme and support on immobilisation were studied. Between 58 and 92% of the added phosphatase was strongly bound to the humates, depending on the conditions of immobilisation used. Some characteristics of the humate–phosphatase complexes and of the free enzyme were compared. The enzymatic complexes showed values of K_m (2.22 mM ) and activation energy (33.4 kJ mol^?1) similar to those of the free enzyme (2.00 mM and 27.6 kJ mol^?1). The pH/activity profiles revealed no change in terms of shape or optimum pH (10.5) upon immobilisation of alkaline phosphatase. However, the immobilised enzyme showed maximal activity in the range of 80–100 °C, while the free enzyme had its highest activity at 60 °C. The thermal stability of alkaline phosphatase was enhanced by complexation to the soil humates. © 2003 Society of Chemical Industry     

Potentiality of Bacillus amyloliquefaciens KFCC11574P isolated from Korean traditional doenjang as a starter in the production of functional soya bean paste

Proteolytic activity, fibrinolytic activity and angiotensin converting enzyme (ACE)‐inhibitory activity, volatile compound profile, off‐flavour compound and sensory characteristics in soya bean paste made with Bacillus amyloliquefaciens KFCC11574P (KHG19) strain were compared with those in three different commercial soya bean paste samples. The KHG19 strain was isolated from doenjang, a traditional Korean soya bean paste, and the maximum dominance in soya bean paste prepared in this study was 68.8% after 2 months of ageing. Proteolytic and fibrinolytic activities in soya bean paste made with KHG19 strain (KHG19 soya bean paste) were higher than those in commercial soya bean pastes. The maximum proteolytic activity (2.47 cm²) was observed after 3 months of ageing, and the maximum relative fibrinolytic activity (236.98%) was appeared after 2 months of ageing. ACE‐inhibitory activity in KHG19 soya bean paste was similar to those in commercial samples. The volatile compound profile of KHG19 soya bean paste was closest to that of cheonggukjang according to the result from principal component analysis. The sensory score for off‐flavour character was significantly low in KHG19 soya bean paste (2.9) compare with the score (6.8) for Korean traditional doenjang. KHG19 strain can be used for the production of functional soya bean paste that would be affordable to global consumer.     

Optimisation of drying conditions for the extraction of β‐carotene,phenolic and ascorbic acid content from yellow‐fleshed sweet potato using response surface methodology

The objective of this study was to optimise drying conditions for the extractions of β‐carotene, phenolic and ascorbic content from yellow‐fleshed sweet potato using response surface methodology. A face‐centred cubic was used to investigate the effects of three independent variables namely drying temperature 55–65 °C, citric acid concentration 1–3%w/v and soaking time 1–3 min. The optimal conditions for parameters were 55–62 °C, citric acid concentration 1.08–2.19% and soaking time 1.53–2.00 min. Under the above mentioned conditions, the experimental β‐carotene content was 14.61 mg g^?1, total phenolic and ascorbic acid content were 4.0 and 17.53 mg g^?1, respectively, which were close to the predicted values. Therefore, the results showed that optimise conditions could be used to enhance the antioxidant activities of functional foods.     

Detoxification of jack beans (Canavalia ensiformis): I.—Extrusion and canavanine elimination

In order to remove canavanine from jack bean (Canavalia ensiformis) the authors conducted extrusion and extraction experiments. In the extrusion experiments, using jack bean without testa and conditioned to 240 g kg^?1 moisture content, different temperatures of the compression section of the extruder and screw rates were tried. Under these conditions the parameters measured were expansion, final moisture, haemagglutinins, urease activity, available lysine and soluble nitrogen index. The screw rate did not affect the extrusion of jack bean. The best temperature was 110°C. The extraction experiments were carried out with extruded jack bean (EJB) at 110°C and 70 rpm using one or two acid or formalin extractions. EJB amount, times and pH were evaluated with a surface response model in one acid extraction experiment. The best results were observed with 100 g kg^?1 of EJB, at pH 55 for 2 h (1AEJB), which reduced the canavanine content to 8.5 g kg^?1. In the same conditions using a second acid extraction (2AEJB) we obtained a product with 4.9 g kg^?1 of canavanine. With the formalin extraction at pH 5.5 and 7.5, and a molar ratio of canavanine: formalin 1 : 1 and 1 : 2, we obtained a product with 8.2 g kg^?1 of canavanine.     

Synthesis and in vitro digestion of resistant starch type III from enzymatically hydrolysed cassava starch

Resistant starch type III (RS III) was synthesised from cassava starch by autoclaving followed by debranching with pullulanase, at varied concentrations (0.4–12 U g^?1) and times (2–8 h), and recrystallisation (?18 to 90 °C for 1–16 h). The highest RS III yield (22 g/100 g) was obtained at an enzyme concentration of 4 U g^?1 after 8 h incubation, followed by recrystallisation at 25 °C for 16 h. Varying the recrystallisation conditions indicated that higher RS III yields (30–35 g/100 g) could be obtained at 90 °C within 2 h. Thinning cassava starch using α‐amylase prior to debranching using pullulanase did not further increase the RS III content. In vitro digestion data showed that whereas 44% RS III was digested after 6 h, the corresponding value for cassava starch was 89%.     

Activated Carbon and Ion Exchange Treatments for Removing Phenolics and Phytate from Peanut Protein Products

Commercial defatted peanut flour and laboratory defatted peanut meal contained 1.5 and 1.7% phytate, 1756 and 2033 μg g^?1 total phenolic acids and 50 and 120 μg g^?1 neutral phenolics, respectively. Laboratory peanut protein isolate contained undetectable amounts of neutral phenolics, 810 μg g^?1 total phenolic acids and about 1% phytate. The ion exchange treatment removed ≥85% of the phytate and the ion exchange and activated carbon treatments removed 92 and 82%, respectively, of the total phenolic acids from peanut protein isolate. p-Coumaric acid was the major phenolic acid, accounting for 40–68% of the total phenolic acids in all peanut protein products.     

Impact of cycling temperatures on Fusarium verticillioides and Fusarium graminearum growth and mycotoxins production in soybean

BACKGROUND: Fusarium graminearum and F. verticillioides are two very important mycotoxigenic species as they cause diverse diseases in crops. The effects of constant and cycling temperatures on growth and mycotoxin production of these species were studied on soybean based medium and on irradiated soya beans. RESULTS: F. graminearum grew better when was incubated at 15, 20 and 15–20 °C (isothermal or cycling temperature) during 21 days of incubation. Maximum levels of zearalenone and deoxynivalenol (39.25 and 1040.4 µg g^?1, respectively) were detected on soya beans after 15 days of incubation and the optimal temperature for mycotoxin production was 15 °C for zearalenone and 20 °C for deoxynivalenol. F. verticillioides grew better at 25 °C in culture medium and at 15/20 °C and 15/25 °C on soybean seeds. Fumonisin B₁ was produced only in culture medium, and the maximum level (7.38 µg g^?1) was found at 15 °C after 7 days of incubation. CONCLUSION: When growth and mycotoxin production under cycling temperatures were predicted from the results under constant conditions, observed values were different from calculated for both species and substrate medium. Therefore, care should be taken if data at constant temperature conditions are to be extrapolated to real field conditions. Copyright © 2012 Society of Chemical Industry     

Instron measurement of sponge cake firmness: Effect of additives and storage conditions

Instron measurement and sensory analysis of sponge cake texture was performed under various conditions. The samples were compared on the basis of their storage in modified atmosphere (CO₂/N₂, 50:50 v) or not, at + 4°C or + 20°C, as whole cakes or slices of cake, and with or without additives (soya bean protein or sodium alginate). It was found that the best conditions for preservation of a soft texture in a whole cake are obtained in a modified atmosphere at +4°C when 10 g kg^?1 soya bean protein is included in the sponge cake formulation.     

Xylitol bioproduction from wheat straw: hemicellulose hydrolysis and hydrolyzate fermentation

A 2² central composite design with five center points was performed to estimate the effects of temperature (120, 130 and 140 °C) and acid loading (100, 150 and 200 mg g^?1) on the yield of monomeric xylose recovery from wheat straw hemicellulose (Y_S/RM). Under the best hydrolysis condition (140 °C and 200 mg g^?1), a Y_S/RM of 0.26 g g^?1 was achieved. After vacuum concentration and detoxification by pH alteration and active charcoal adsorption, the hydrolyzate was used as source of xylose for xylitol bioproduction in a stirred tank reactor. A xylitol production of 30.8 g L^?1 was achieved after 54 h^?1 of fermentation, resulting in a productivity (Q_P) of 0.57 g L^?1 h^?1 and bioconversion yield (Y_P/S) of 0.88 g g^?1. The maximum specific rates of xylose consumption and xylitol production were 0.19 and 0.15 g g^?1 h^?1, respectively. Copyright © 2006 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF HOMOGENEOUS ACID PHOSPHATASE FROM NONGERMINATED BUCKWHEAT (FAGOPYRUM ESCULENTUM) SEEDS

A buckwheat acid phosphatase (orthophosphoric‐monoester phosphohydrolase, EC 3.1.3.2) was purified about 250‐fold from nongerminated buckwheat seeds to apparent homogeneity with a recovery of 4% from the acid phosphatase activity in the crude extract. It is the major acid phosphatase among eight different acid phosphatases identified in the crude extract. The purified enzyme behaved as a monomeric protein of molecular mass about 45 kDa. The purified enzyme exhibited a single pH optimum at 5.25. Optimum temperature for the degradation of p‐nitrophenyl phosphate was 50C. The kinetic parameters for the hydrolysis of p‐nitrophenyl phosphate were determined to be K_M= 76 μmol L^?1 and k_cat= 924 s^?1 at pH 5.25 and 37C. While the enzyme failed to act on phytate as a substrate, the enzyme exhibited a broad substrate selectivity. The purified enzyme showed no measureable carboxylesterase activity and no divalent metal ion requirement.     

α-Amylase inhibitor levels in seeds generally available in Europe

The α-amylase inhibitor (αAI) levels in 18 seeds, which are generally available throughout Europe, have been determined. Kidney beans, haricot beans, pinto beans and runner beans had high contents of αAI (2–4 g equivalent kg^?1 seed meal). Butter beans, blackeyed peas, chickpeas, field beans and sweet lupinseeds contained 0.1–0.2 g inhibitor equivalent kg^?1 seed meal. No αAI activity was detected in samples of adzuki bean, lentils, mung beans, peas, soya beans, sunflower seeds or winged beans. The αAI activity present in whole kidney beans was relatively heat-resistant. However, it could be completely abolished by aqueous heat treatment of fully imbibed beans at 100°C for 5–10 min.     

Protein concentrate from Jatropha curcas screw‐pressed seed cake and toxic and antinutritional factors in protein concentrate

BACKGROUND: Jatropha curcas seeds are highly toxic to livestock. The presence of phorbol esters and antinutrients such as trypsin inhibitor, lectin and phytate and the high level of shells in the seed cake prevent its use in animal diets. Using the principle of isoelectric precipitation, the conditions for preparation of the protein concentrate from oil‐containing seed cake and defatted seed cake were optimised and the contents of phorbol esters and antinutrients were determined. RESULTS: The recovery of protein concentrate was highest when the proteins from the seed cakes were solubilised at pH 11 for 1 h at 60 °C and the precipitation of these proteins was done by lowering the pH to 4. Under these conditions, over 53% of the total proteins present in the seed cakes were recovered in the protein concentrates. The protein contents in the protein concentrates obtained from the oil‐containing seed cake and defatted seed cake were 760 and 820 g kg^?1 respectively. Substantial amounts of phorbol esters were present in the protein concentrates (0.86–1.48 mg g^?1). Trypsin inhibitor was present at an approximately tenfold higher level in the protein concentrates than in the seed cakes. Lectin and phytate were also present at high levels, but their levels were lower than in the seed cakes. Tannins were present in negligible amounts. CONCLUSION: To make the protein concentrate from Jatropha seed cake fit for use as an ingredient in livestock feed, phorbol esters must be removed and trypsin inhibitor and lectin inactivated by heat treatment. The adverse effects of phytate could be mitigated by addition of phytase in the diet. Copyright © 2008 Society of Chemical Industry