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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.

Abstract The aim of the present study was to fabricate, characterize, and evaluate the in vitro antimicrobial and antioxidant properties of zein/polyvinyl alcohol (ZN/PVA) nanofibers containing 2% and 4% of thymoquinone (TQ), either alone or in combination with electrosprayed ZN nanoparticles containing 1% and 2% of resveratrol (RS). According to scanning electron microscopy analysis, the diameter of nanofibers and nanoparticles increased with increasing TQ and RS concentrations, respectively. The molecular interaction between ZN or PVA polymers and TQ or RS was confirmed by Fourier transform infrared spectroscopy. Thermogravimetric analysis showed that the thermal stability of nanofibers did not change with the addition of TQ and RS. Moreover, incorporation of TQ in nanofibers along with RS nanoparticles increased their antibacterial and free radical scavenging activities based on broth dilution and DPPH methods, respectively (p ≤ .05). Escherichia coli O157:H7 (as a Gram‐negative pathogenic bacteria) was more resistant to all treatments than Staphylococcus aureus (as a Gram‐positive pathogenic bacteria). In addition, the combined use of TQ in nanofibers and RS nanoparticles had antagonistic antibacterial and synergistic antioxidant effects. The best results were obtained with ZN/PVA nanofiber containing 4% TQ and electrosprayed with 2% RS nanoparticles (p ≤ .05). According to the results of the present study, biodegradable ZN/PVA nanofiber containing TQ and electrosprayed with RS nanoparticles can be used as a novel active packaging material in the food industry.

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.

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.

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.

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.

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.