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Abstract The food enzyme β‐glucosidase (β‐d‐glucoside glucohydrolase, EC 3.2.1.21) is produced with the non‐genetically modified Penicillium guanacastense strain AE‐GLY by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in four food manufacturing processes. Subsequently, the applicant has requested to extend its use to include three additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. The dietary exposure was calculated to be up to 0.206 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Using the no observed adverse effect level reported in the previous opinion (943 mg TOS/kg bw per day), the Panel derived a margin of exposure of at least 4578. Based on the previous evaluation, the assessment of the new data and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The food enzyme carboxypeptidase C (EC 3.4.16.5) is produced with the genetically modified Aspergillus niger strain PEG by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in nine food manufacturing processes. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 2.053 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with a xylanase obtained from A. niger strain XEA. The Panel considered this food enzyme as a suitable substitute for the carboxypeptidase to be used in the toxicological studies, because both strains were derived from the same recipient strain, the location of the inserts was comparable, no partial inserts were present and the production methods were essentially the same. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1850 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 901. A homology search for the amino acid sequence of the food enzyme to known allergens was made and one match with a wheat allergen was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially in wheat‐allergic individuals, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme has four declared activities: endo‐polygalacturonase ((1–4)‐α‐d‐galacturonan glycanohydrolase (endo‐cleaving); EC 3.2.1.15), pectinesterase (pectin pectylhydrolase; EC 3.1.1.11), pectin lyase ((1–4)‐6‐O‐methyl‐α‐d‐galacturonan lyase; EC 4.2.2.10) and non‐reducing end α‐l‐arabinofuranosidase (α‐l‐arabinofuranoside non‐reducing end α‐l‐arabinofuranosidase; EC 3.2.1.55). It is produced with the non‐genetically modified Aspergillus niger strain PEC by DSM Food Specialties B.V. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant has requested to extend its use to include four additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. As the food enzyme–total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme–TOS was estimated only for the remaining six processes. The dietary exposure was calculated to be up to 0.612 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (204 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 333. Based on the previous evaluation, the assessment of the new data and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The food enzyme endonuclease (Aspergillus nuclease S1; EC 3.1.30.1) is produced with the non‐genetically modified Penicillium citrinum strain NP 11–15 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.006 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1010 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 168,333. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially for individuals allergic to Penicillium. However, the likelihood of such reactions will not exceed the likelihood of allergic reactions to Penicillium. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non‐genetically modified Aspergillus tubingensis strain NL151 by Shin Nihon Chemical Co., Ltd. The food enzyme was free from viable cells of the production organism. It is intended to be used in six food manufacturing processes. Dietary exposure was estimated to be up to 0.278 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1669 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 6004. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract The food enzyme, a triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3), is produced with the non‐genetically modified Limtongozyma cylindracea strain AE‐LAYH (B) by Amano Enzyme Inc. It is intended to be used in six food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining five food manufacturing processes. It was estimated to be up to 0.315 mg TOS/kg body weight (bw) per day in European populations. As the production strain qualifies for the quality presumption of safety (QPS) approach of safety assessment and no issue of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary. A homology search for the amino acid sequence of the food enzyme to those of known allergens was made and one match with a honeybee venom allergen was found. The Panel considered that a risk of allergic reactions by dietary exposure, particularly in individuals allergic to honey, cannot be excluded, but is considered to be low. Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the food enzyme manufacturing process, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme endo‐1,3(4)‐β‐glucanase (3‐(1–3;1–4)‐β‐d‐glucan 3(4)‐glucanohydrolase; EC 3.2.1.6) is produced with the non‐genetically modified Talaromyces versatilis strain PF8 by Erbslöh Geisenheim AG. The food enzyme was free from viable cells of the production organism. It is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme–total organic solids (TOS) was calculated to be up to 0.110 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 2229 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure resulted in a margin of exposure of at least 20,264. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and four matches with respiratory or contact allergens were found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract The food enzyme β‐galactosidase (β‐d‐galactoside galactohydrolase; EC 3.2.1.23) is produced with the genetically modified Bacillus licheniformis strain DSM 34099 by Kerry Group Services International, Ltd. (KGSI). The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is intended to be used in two food manufacturing processes. Dietary exposure was estimated to be up to 7.263 mg total organic solids/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests, other than an assessment of allergenicity, were considered unnecessary by the Panel. A search for the identity of the amino acid sequence of the food enzyme to known allergens was made and one match with a food allergen from kiwi fruit was found. The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to kiwi fruit, cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract The food enzyme glucan 1,4‐α‐maltohydrolase (4‐α‐d‐glucan α‐maltohydrolase; EC 3.2.1.133) is produced with the genetically modified Saccharomyces cerevisiae strain LALL‐MA+ by Danstar Ferment AG. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the processing of cereals and other grains for production of baked products. Dietary exposure was estimated to be up to 0.014 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the identity of the amino acid sequence of the food enzyme to known allergens was made and four matches were found, three with respiratory allergens and one with an allergen from mosquito (injected). The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non‐genetically modified Rhizopus arrhizus strain AE‐TL(B) by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in two food manufacturing processes. Subsequently, the applicant requested to extend its use to include four additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of six food manufacturing processes. As the food enzyme‐total organic solids (TOS) are removed from one food manufacturing process, the dietary exposure to the food enzyme‐TOS was estimated only for the remaining five processes. Dietary exposure was calculated to be up to 0.086 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level reported in the previous opinion (1960 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 22,791. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The food enzyme thermolysin (EC. 3.4.24.27) is produced with the non‐genetically modified Anoxybacillus caldiproteolyticus strain AE‐TP by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in eight food manufacturing processes. Subsequently, the applicant has requested to extend its use to one additional process, to withdraw two processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of seven food manufacturing processes. The dietary exposure to the food enzyme–total organic solids (TOS) was calculated to be up to 0.989 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level reported in the previous opinion (700 mg TOS/kg bw per day, the mid‐dose tested), the Panel derived a revised margin of exposure of at least 708. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase i.e. EC 3.2.1.1) is produced with the non‐genetically modified Cellulosimicrobium funkei strain AE‐AMT by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that the food enzyme did not give rise to safety concerns when used in seven food manufacturing processes. Subsequently, the applicant has requested to extend its use to include three additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of ten food manufacturing processes. As the food enzyme‐total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme‐TOS was estimated only for the remaining nine processes. The dietary exposure was calculated to be up to 0.049 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (230 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 4694. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Abstract The food enzyme pullulanase (pullulan 6‐α‐glucanohydrolase; EC 3.2.1.41) is produced with the non‐genetically modified Pullulanibacillus naganoensis strain AE‐PL by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in one food manufacturing process. Subsequently, the applicant has requested to extend its use to include seven additional processes and to revise the previous use level. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of eight food manufacturing processes. As the food enzyme‐total organic solids (TOS) are not carried into the final foods in two food manufacturing processes, the dietary exposure was estimated only for the remaining six processes. The dietary exposure was calculated to be up to 0.004 mg TOS/kg body weight (bw) per day in European populations. The Panel evaluated the repeated dose 90‐day oral toxicity study in rats submitted in the previous application and identified a no observed adverse effect level of 643 mg TOS/kg bw per day, the highest dose tested. When compared with the calculated dietary exposure, this resulted in a margin of exposure of at least 160,750. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the non‐genetically modified Aspergillus luchuensis strain AE‐L by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in one food manufacturing process. Subsequently, the applicant has requested to extend its use to include four additional processes and to revise the previous use level. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of five food manufacturing processes. The dietary exposure to the food enzyme‐total organic solids (TOS) was calculated to be up to 0.458 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1726 mg TOS/kg bw per day, the highest dose tested), the Panel derived a revised margin of exposure of at least 3769. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Abstract The food enzyme lysophospholipase (2‐lysophosphatidylcholine acylhydrolase, EC 3.1.1.5) is produced with the genetically modified Trichoderma reesei strain DP‐Nyc81 by Genencor International B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the processing of cereals and other grains for the production of glucose syrups and other starch hydrolysates. Since residual amounts of food enzyme–total organic solids are removed during these food manufacturing processes, dietary exposure was not calculated and toxicological studies were considered unnecessary. A search for the identity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract The food enzyme oryzin (EC 3.4.21.63) is produced with the non‐genetically modified Aspergillus ochraceus strain AE‐P by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in nine food manufacturing processes. Subsequently, the applicant has requested to extend its use to one additional process, to withdraw two food processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of eight food manufacturing processes. The dietary exposure to the food enzyme‐total organic solids (TOS) was calculated to be up to 0.354 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level reported in the previous opinion (1862 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 5260. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Breast milk is the main source of nutrition during early life, but both infant formulas (Ifs; up to 12 months) and baby foods (BFs; up to 3 years) are also important for providing essential nutrients. The infant food industry rigorously controls for potential physical, biological, and chemical hazards. Although thermal treatments are commonly used to ensure food safety in IFs and BFs, they can negatively affect sensory qualities, reduce thermosensitive nutrients, and lead to chemical contaminant formation. To address these challenges, non-thermal processing technologies such as high-pressure processing, pulsed electric fields, radio frequency, and ultrasound offer efficient pathogen destruction similar to traditional thermal methods, while reducing the production of key process-induced toxicants such as furan and 5-hydroxymethyl-2-furfural (HMF). These alternative thermal processes aim to overcome the drawbacks of traditional methods while retaining their advantages. This review paper highlights the growing global demand for healthy, sustainable foods, driving food manufacturers to adopt innovative and efficient processing techniques for both IFs and BFs. Based on various studies reviewed for this work, the application of these novel technologies appears to reduce thermal processing intensity, resulting in products with enhanced sensory properties, comparable shelf life, and improved visual appeal compared to conventionally processed products.

This research aimed at analyzing the phytase from fungal isolates SB1, SB2, BS, and WF produced in cornstarch with glucose medium (CS+Glu) as carbon sources and Potatoes dextrose broth (PDB). The activity of phytase from isolates SB1, SB2, BS, and WF produced in CS+Glu medium was 2.97 UmL-1, 2.87 UmL-1, 3.18 UmL-1, and 4.37 UmL-1, respectively, while the activity of phytases was 2.07 UmL-1, 2.17 UmL-1, 2.22 UmL-1, and 2.78 UmL-1 respectively in PDB medium. The optimal temperature of SB1 and WF phytase was 40°C, while SB2 and BS were 50°C and 60°C, respectively. The optimal pH of SB1 and WF phytase was 5.0, while SB2 and BS phytase were 6.0, and 4.0 respectively. 18S rRNA gene analysis revealed that SB1 was 99% identical to Aspergillus niger ANTS (KY825168.1), SB2, BS, and WF were 99% identical to A. niger Moriga leaf (MG889596.1). Multiple sequences and phylogenetic analysis of phytase gene showed that phyA_SB1 and phyA_SB2 were 98% homology with A. ficuum (AAB26466), 97% with A. niger (ADP05107) while phyA_WF was 99% with A. ochraceoroseus (PLB29348), 98% with A. niger (ADP05105). The deduced proteins contain conserved motifs RHGARYPTD at N-terminal while lacking HD motif at C-terminal. These phytases were in the same cluster with Aspergillus sp. phytase A indicating that they belong to Histidine Acid Phosphatases (HAP) family.

Grass pea (Lathyrus sativus L.) is commonly consumed in cooked, fermented, and roasted forms in Ethiopia. However, the impacts of household processing practices on its nutrients, antinutrients, and toxic compounds have not been adequately studied. Therefore, the effects of household processing and fermentation in the presence and absence of a phytase on the contents of β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), myo-inositol phosphates, crude protein, minerals and the in vitro bioaccessibility were investigated. Fermentation exhibited a significant decline in β-ODAP (13.0–62.0%) and phytate (7.3–90.5%) irrespective of the presence of phytase. Pressure and pan cooking after discarding the soaking water resulted in a 27.0 and 16.2% reduction in β-ODAP. A 30% reduction in phytate was observed during germination followed by roasting. In addition, germination resulted in a significant (p < 0.05) increase in crude protein. Germination and germination followed by roasting resulted in the highest Fe bioaccessibilities (more than 25 fold higher compared to untreated samples) followed by pressure cooking and soaking. Processing also improved Zn bioaccessibilities by 50.0% (soaked seed without soaking water), 22.5% (soaked seed with soaking water), and 4.3% (germination). Thus, the processing technologies applied were capable of reducing the content of phytate (InsP6) and β-ODAP with a concomitant increase in mineral bioaccessibilities. Processing of grass peas could therefore contribute to their more widespread utilization.

The aim of this work was to substantiate the use of whey protein extract (WPE), a by-product of dairy industry, combined with different berry purees in the formulation of a whipped frozen dairy dessert (WFDD). After freezing, the swelling ability of WPE decreased significantly while at higher temperatures of the dispersion medium, the degree of swelling increased. A mixture containing 55±2 % WPE and 45±2 % milk presented a homogeneous consistency while the further processing did not improve the structure of the mixture. Final formulations of WFDD containing different berry purees (i.e. 10 % black¬currant puree, 25 % cherry puree, or 30 % strawberry puree) were proposed for which the nutritional facts were calculated. Recommended shelf life of the developed desserts at -18 °C is suggested to be 20 days. Regulatory requirements and recommendations for the production of WFDD in an industrial scale were also developed. Recommended shelf life of the developed desserts at -18 °C is suggested to be 20 days. In conclusion, a WFDD was developed using WPE as a by-product of dairy industry.

Plant pathogens translocate type III effector (T3E) proteins that may be recognized by plants to trigger immunity. Here, the authors show that the Xanthomonas T3E XopH possesses a novel 1-phytase activity that is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene.

Aquaponics are efficient systems that associate aquatic organisms’ production and plants by recirculating water and nutrients between aquaculture and hydroponic tanks. In this study, we characterised the bacterial communities in the freshwater aquaponics system that can mineralise polysaccharides and phytate by producing carbohydrate-degrading enzymes and phytases, by 16S rRNA gene sequencing and in vitro culture techniques. Around 20% of the operational taxonomic units (zOTUs) identified were previously reported to carry fibre-degrading enzyme putative genes, namely β-glucanase (1%), xylanase (5%), or cellulases (17%). Ten % of the zOTUs were previously reported to carry putative genes of phytases with different catalytic mechanisms, namely β-propeller (6%), histidine acid phytases (3%), and protein tyrosine phytase (Staphylococcus sp. and Rossellomorea sp. could produce consistent extracellular phytate-degrading activity. The PCR amplification of β-propeller genes both in environmental samples and in the isolates obtained showed that this is the most ecologically relevant phytase type in the aquaponics systems used. In summary, the aquaponics system is abundant with bacteria carrying enzymes responsible for plant-nutrient mineralisation.

The production of phytase in a recombinant E.coli using the pBAD expression system was optimized using response surface methodology with full-factorial faced centered central composite design. The ampicilin and arabinose concentration in the cultivation media and the incubation temperature were optimized in order to maximize phytase production using 2 3 central composite experimental design. With this design the number of actual experiment performed could be reduced while allowing eludidation of possible interactions among these factors. The most significant parameter was shown to be the linear and quadratic effect of the incubation temperature. Optimal conditions for phytase production were determined to be 100 µg/ml ampicilin, 0.2 % arabinose and an incubation temperature of 37ºC. The production of phytase in the recombinant E. coli was scaled up to 100 ml and 1000 ml.

ABSTRACTIn this study, 154 isolates capable of producing extracellular phytate-degrading activity were isolated from four soil samples from volcanic areas in Central Java, Indonesia. Six strains with high phytate-degrading activity were selected for strain identification and characterization of the corresponding phytate-degrading enzyme. Blast analysis of 16S rRNA gene sequences revealed high similarities for all the six isolates to reference sequences belonging to the genusBacillus. Isolates MS5, MC6, D10 and D16 showed 99% sequence identity toB. cereus, while isolate MC8 exhibited 99% sequence identity toB. aryabhatti and D6 99% sequence identity toB. psychrotolerans. The crude extracellular phytase preparations from the isolates showed following optimal conditions for phytate dephosphorylation: pH 4.0 and 50°C (isolate D10), pH 5.0 and 60°C (isolate MC6, and isolate MS5), pH 6.0 and 50°C (isolate D16) and pH 6.0 and 60°C (isolate D6) and pH 6.0 and 40°C (isolate MC8). Zn2+ and Fe3+ strongly inhibited phytate dephosphorylation with all phytase preparations studied. In the presence of Ca2+, an increase in phytase activity of 10-15% was obtained.

The production of phytase in a recombinant E.coliusing thepBAD expression system wasoptimized using response surface methodology with full-factorial faced centered central composite design. The ampicilin and arabinose concentrationin the cultivation media and the incubation temperature were optimized in order to maximize phytase productionusing 23 central composite experimental design. With this design the number of actual experiment performed could be reduced while allowing eludidation of possible interactions among thesefactors. The most significant parameter was shown to be the linear and quadratic effect of the incubation temperature. OPtim al conditionsfor phytase production were determinedto be100 μg/ml ampicilin, 0.2 % arabinose and an incubation temperature of37ºC.The production of phytase in the recombinant E. coliwas scaledup to 100ml and 1000ml.

Ferulic acid (FA) and tocopherol (Toc) loaded solid lipid nanoparticles (SLN) were prepared by a hot homogenisation method. The particle size distribution, zeta potential and melting behaviour of the SLN as well as the stability, encapsulation efficiency and radical scavenging activity of FA and Toc in the SLN were analysed. The different formulations containing up to 2.8 mg g-1 of FA or Toc were stable during at least 15 weeks of storage at room temperature. Despite partial degradation and / or release of FA and Toc during storage, significant radical scavenging activity was maintained. DSC measurements and radical scavenging tests after different time periods revealed that the re-structuring of the lipid matrix was connected to the enhanced antioxidant activity of Toc but did not affect the activity of FA.

Nanoparticles are ubiquitous in the environment. They originate from anthropogenic or natural sources or they are intentionally produced for different purposes. There exist manifold applications of nanoparticles in modern life leading unavoidably to a confrontation and interaction between nanomaterial and living organisms. Based on their wide distribution tending to increase steadily, the influence of particles based on silica and silver, exhibiting nominal sizes between 0.65 nm and 200 nm, on the physiology of the mycotoxigenic filamentous fungus Penicillium verrucosum was analyzed. The applied concentration and time-point, the size and the chemical composition of the particles was shown to have a strong influence on growth and mycotoxin biosynthesis. On microscopic scale it could be shown that silver nanoparticles attach to the mycelial surface. Moreover, silver nanoparticles with 0.65 nm and 5 nm in size were shown to internalize within the cell, form agglomerates in the cytoplasm and associate to cell organelles.

Strong increase in the production of genetically modified organisms (GMOs) observed over the years has led to a consolidation of transgenic seed industries worldwide. The dichotomy between the evaluated risk and the perceived risk of transgenic use has defined their level of acceptability among different global societies. GMOs have been widely applied to agricultural commodities, among them the Roundup Ready™ (RR™) soybean line GTS 40-3-2 has become the most prevalent transgenic crop in the world. This variety was developed to confer plant tolerance against glyphosate-based agricultural herbicide Roundup Ready™. Issues related to detection and traceability of GMOs have gained worldwide interest due to their increasing global diffusion and the related socioeconomic and health implications. Also, due to the widespread use of GMOs in food production, labelling regulations have been established in some countries to protect the right of consumers and producers. Besides regulatory demand, consumer concern issues have resulted in the development of several methods of detecting and quantifying foods derived from genetically engineered crops and their raw materials. Polymerase chain reaction (PCR) has been proven to be the method of choice to detect the presence or absence of the introduced genes of GMOs at DNA level. The present paper aims to verify whether the PCR technique can detect RR™ soybean seeds among conventional ones to further certification as non-GM soybean seeds and grains. This analysis could be accomplished through the development of new methodology called 'intentional contamination' of soybean conventional seeds or grains with the respective RR™ soybeans. The results show that the PCR method can be applied with high sensitivity in order to certify conventional soybean seeds and grains.

Micronutrient deficiencies are a major public health problem. Beans are an important plant-based source of iron, zinc and copper, but their absorption is reduced in the presence of anti-nutrients such as phytates, polyphenols and tannins. Soaking and discarding the soaking water before cooking is unanimously recommended, but this can result in mineral loss. Data on the consequences for mineral bioaccessibility is still limited. This study aimed to evaluate iron, zinc and copper bioaccessibility in black beans cooked (regular pan, pressure cooker) with and without the soaking water. For that, three batches of black beans were investigated in triplicate, each split in nine parts (raw grains and four different household processes in duplicate) and analyzed by applying the quarter technique, resulting in a grand total of 164 samples. Minerals were quantified by ICP-MS (inductively coupled plasma mass spectrometry), myo-inositol phosphates (InsP5, InsP6) by HPLC (high-performance liquid chromatography) ion-pair chromatography, total polyphenols using Folin-Denis reagent and condensed tannins using Vanillin assay. Mineral bioaccessibility was determined by in vitro digestion and dialysis. All treatments resulted in a statistically significant reduction of total polyphenols (30%) and condensed tannins (20%). Only when discarding the soaking water a loss of iron (6%) and copper (30%) was observed, and InsP6 was slightly decreased (7%) in one treatment. The bioaccessibility of iron and zinc were low (about 0.2% iron and 35% zinc), but copper presented high bioaccessibility (about 70%). Cooking beans under pressure without discarding the soaking water resulted in the highest bioaccessibility levels among all household procedures. Discarding the soaking water before cooking did not improve the nutritional quality of the beans.

The glucose-1-phosphatase encoding gene (agp) of Pantoea agglomerans was sequenced and heterologously expressed in Escherichia coli. The enzyme showed very high homology to periplasmatic glucose-1-phosphatases of other members of the Enterobacteriaceae family. It was isolated from transformed Escherichia coli cells in a single step in high yields (32.3 ± 1.2 mg per litre of culture) by Ni-NT agarose affinity chromatography to >95% purity as calculated from specific activity determinations. The purified glucose-1-phosphatase was entrapped in alginate beads with an entrapment efficiency of >80%. Temperature stability was enhanced as a consequence of entrapment, whereas pH dependence of enzyme activity was not affected. Maximum catalytic activity of entrapped glucose-1-phosphatase was found at 70°C, whereas the free enzyme exhibited maximal activity at 60°C. A single pH optimum at pH 4.5 was determined for the free and the entrapped enzyme. Kinetic parameters for the hydrolysis of sodium phytate were found to be affected by entrapment. They were determined to be K M = 0.84 mmol l-1 and k cat = 8 s-1 at pH 4.5 and 37°C for the entrapped glucose-1-phosphatase and K M = 0.35 mmol l-1 and k cat = 20.5 s-1 for the free enzyme. Complete conversion of phytate into one single myo-inositol pentakisphosphate isomer, identified as D-myo-inositol(1,2,4,5,6)pentakis-phosphate, was shown to be feasible by using the enzyme-loaded alginate beads in batch operations. The entrapped enzyme showed a high operational stability by retaining almost full activity even after ten uses.

Phytases have important applications in human and animal nutrition because they hydrolyze the phytate present in legumes, cereal grains and oil seeds. This results in an increased availability of minerals, trace elements and amino acids as well as phosphate. Fifty potential phytase-producing fungal strains were isolated from a fertile soil obtained from the northern part of Paraná State in Brazil and other alternative sources using a selective media. Thereafter phytase production was evaluated in solid-state fermentation using different residues from the agroindustry supplemented with a nitrogen source at 60 % of moisture after 96 hours at 30 °C. The highest phytase activity (51.53 units per gram of dry substrate, U/g) was achieved with citric pulp and the soil isolate FS3 in solid-state fermentation. Furthermore, treatment of the substrates prior to fermentation in order to reduce microbial contamination was shown to affect phytase production during solid-state fermentation. Heat treatment resulted in an increase of the concentration of inorganic phosphate, a well known repressor of microbial phytase production, and therefore in a reduction of phytase production. UV exposure of the substrate was shown to reduce microbial contamination without affecting phytase production.

Phytase [myo-inositol(1,2,3,4,5,6)hexakisphosphate phosphohydrolase], a phytate-specific phosphatase, is already used as a supplement in diets for monogastric animals to improvephosphate utilisation from phytate[myoinositol(1,2,3,4,5,6)hexakisphosphate], the major storage form of phosphate in plant seeds. In recent years, this class of enzymes has also been found increasingly interesting for use in processing and manufacturing of food for human consumption, particularly because the decline in food phytate results in an enhancement of mineral bioavailability. Different strategies could be applied to optimise phytate degradation during food processing and digestion in the human alimentary tract such as adjustment of more favourable conditions during food processing for the phytases naturally occurring in the raw material, addition of isolated phytases to the production process, use of raw material with a high intrinsic phytate-degrading activity either naturally present or introduced by genetic engineering and the use of recombinant food-grade microorganisms as carriers for phytate-degrading activity in the human gastrointestinal tract. Furthermore, phytases may find application in the production of functional foods or food supplements with health benefits. Last but not least, technological improvements are expected to occur due to phytate degradation during processing as shown for breadmaking, production of plant protein isolates, corn wet milling and the fractionation of cereal bran.

Abstract  Over two hundred bacteria were isolated from the halosphere, rhizosphere and endophyte of Malaysian maize plantation and screened for phytases activity. Thirty isolates with high detectable phytase activity were chosen for media optimization study and species identification. Ten types of bacterial phytase producers have been discovered in this study, which provides opportunity for characterization of new phytase(s) and various commercial and environmental applications. The majority of the bacterial isolates with high detectable phytase activity were of endophyte origin and 1.6% of the total isolates showed phytase activity of more than 1 U/ml. Most of the strains produced extra-cellular phytase and Staphylococcus lentus ASUIA 279 showed the highest phytase activity of 1.913 U/ml. All 30 species used in media optimization study exhibit favorable enzyme production when 1% rice bran was included in the growth media. [ABSTRACT FROM AUTHOR]

Abstract  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. [ABSTRACT FROM AUTHOR]